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NAD(P)H:quinone oxidoreductase 1 reduces the mutagenicity of DNA caused by NADPH:P450 reductase-activated metabolites of benzo(a)pyrene quinones.

The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was studied using supF tRNA gene as the mutational target. pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6). Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations. P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations. Oxygen radical scavengers (SOD and catalase) blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations. This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q. Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine. Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed. Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations. However, BP-hydroquinone (BP-3,6-HQ) generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' in supF tRNA gene at a significantly high frequency. A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds. These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS).

Another important class of BP metabolites generated during its oxidative metabolism are the benzo(a)pyrene quinones (BP-3,6-Q, BP-1,6-Q and BP-6,12-Q) (Gelboin, 1980;O'Brien, 1991). Quinones, in general, are ubiquitously found in all aerobic plants and animals and are present in the environment as part of automobile exhaust, cigarette smoke and urban air particulates (O'Brien, 1991;Monks et al, 1992). Several compounds containing the quinone nucleus are extensively used as anti-tumour drugs (Workman, 1994). Because of their electrophilic nature and the very high redox potential, the quinones are highly toxic (O'Brien, 1991;Monks et al, 1992;Workman, 1994). Despite being present in significant amounts, thus posing a potential threat for extensive human exposure, and being highly reactive and toxic, the mechanisms underlying the toxicity of quinones, particularly with respect to their mutagenic and carcinogenic potential, are rather poorly understood. Earlier studies have shown that the fate of quinones inside the cells depends on the balance between phase I (cytochromes P450 and P450 reductase) enzymes that catalyse metabolic activation of quinones, leading to cellular damage, and phase II [NAD(P)H:quinone oxidoreductase 1, glutathione Stransferase and UDPG transferase) enzymes that catalyse metabolic detoxification of quinones, resulting in protection of the cells (Talalay et al, 1995). Accordingly, it has been proposed that induction of phase II enzymes by antioxidants, vitamins, isothiocyanates and related compounds leads to protection of cells against adverse effects of exposure to quinones and their precursors (Prestera et al, 1993;Zhang et al, 1994;Talalay et al, 1995). Recent studies in our laboratory on metabolic activation and detoxification of BPQs (BP-3,6-Q, BP-1,6-Q and BP-6,12-Q) provided evidence at the molecular level in support of the above hypothesis . Accordingly, the one-electron reduction of BPQ by P450 reductase resulted in the generation of benzo(a)pyrene semiquinones (BPSQ) and reactive oxygen species (ROS). The metabolites of BPQ (BPSQ and ROS) bind with the deoxyguanosine residues of the DNA, as evident from the generation of BPQ (metabolites)-DNA adducts. On the other hand, NQOl competed with P450 reductase and catalysed two-electron reduction and detoxification of BPQ, thus protecting the cells from the generation of BPSQ, ROS and the corresponding DNA adducts.
In the present report, we have extended our studies on the mutagenicity of the unmetabolized quinones and P450 reductaseactivated quinones (semiquinones and ROS) using BP-3,6-Q as the model quinone and using the supF suppressor tRNA gene of the E. coli plasmid pUB3 as the mutational target to understand the genotoxic basis for the mutagenic and carcinogenic potential of quinones. We also extended our studies to determine whether NQOl exclusively reduces/prevents the mutagenicity of semiquinone and ROS or whether it also stimulates mutagenicity of quinones. The various results obtained by us demonstrate that P450 reductase-activated BPQ (BPSQ and ROS) binding to the DNA results in mutations (predominantly base deletions and substitutions). We also demonstrate that these mutations are caused as a result of the binding of ROS to the DNA. We further demonstrate that NQOl reduces/prevents the mutations caused by P450 reductase-activated BPQ. In addition, we demonstrate that NQO1 catalyses the conversion of BPQ to BP hydroquinone (BPHQ), which causes specific mutations involving deletion of a single cytosine from the sequence 5'-CCCCC-3' at a very high frequency, which was not observed with BPQ and P450 reductase-activated BPQ (BPSQ and ROS). We further demonstrate that deletion of a single cytosine from the sequence 5'-CCCCC-3' is specifically associated with the hydroquinone class of compounds. This is because, in addition to BP-3,6-HQ, benzoquinone hydroquinone (HQ) also causes similar mutations at high frequency. The quantitative and qualitative aspects of mutations due to BPQ as well as its one-and two-electron reductive metabolites are compared with those due to BPDE and the possible implications are discussed.

MATERIALS AND METHODS Materials
The E. coli strain ES87 and the plasmid pUB3 carrying the mutational target supF suppressor tRNA were obtained as a generous gift from Dr E Loechler (Boston University, Boston, MA, USA). DH5-a cells were purchased from Gibco-BRL (Gaithersburg, MD, USA). Ingredients of the media used to grow the bacterial cells and to select the mutants were purchased from Difco Laboratories (Detroit, MI, USA). All other reagents used in the experiments were of the highest purity available commercially.
The DNA sequencing kit version 2.0 was purchased from USB Corporation, Cleveland, OH, USA. +(-)anti-BPDE and BP-3,6quinone were purchased from the Chemical Carcinogen Repository of the National Cancer Institute (Kansas City, MO, USA). Benzoquinone hydroquinone (HQ) was purchased from Sigma Chemical Company, St Louis, MO, USA.
Construction of pMT2-cDNA recombinant plasmids, transient transfection and enzyme assays Human cDNAs encoding microsomal P450 reductase and cytosolic NQO1 have been cloned and sequenced (Jaiswal et al, 1988;Yamano et al, 1989). cDNAs for P450 reductase and NQOI were separately subcloned into the unique EcoRl site of the expression vector pMT2 (Shaw et al, 1991;Joseph and Jaiswal, 1994). The pMT2, pMT2-P450 reductase and pMT2-NQO1 plasmids were transiently transfected into monkey kidney COS1 cells in separate experiments by the DEAE-dextran and chloroquine method to overexpress the respective proteins/activity (Shaw et al, 1991;Joseph and Jaiswal, 1994). The transfected COS1 cells were harvested, homogenized and subcellular-fractionated by procedures as described . The whole homogenate and microsomal and cytosolic fractions were analysed for P450 reductase and NQOI activities using methods as described . Protein contents in the various fractions were estimated using Bradford's method (Bradford, 1976). The cytosolic and microsomal fractions of transfected COS I cells were used as the source of P450 reductase and NQO1 enzymes, respectively, in incubation mixtures to study the role of these enzymes in metabolic reductive activation or detoxification of BP-3,6-Q, as determined by analysis of mutations in the supF tRNA region of plasmid pUB3.
ES87 cells and pUB3 system to study mutations caused by BPDE and BP-3,6-Q. Adduction of pUB3 DNA with BPDE, BP-3,6-Q and hydroquinone pUB3 plasmid DNA, free from RNA and protein, was prepared using the Qiagen plasmid preparation kit (Qiagen, CA, USA) and further purified using phenol-chloroform extraction and ethanol precipitation (Sambrook et al, 1989). For the DNA adduct formation, 10 ,ug of pUB3 DNA was incubated with 100 ng of BPDE in a total volume of 200 ,ul of Tris buffer, pH 7.4, in the dark for 2 h. In the case of BP-3,6-Q-pUB3 DNA adducts, 1O gM BP-3,6-Q and 10 jg of the plasmid DNA were incubated either with the cytosolic NQOl (15 ,ug of total protein) or with the microsomal P450 reductase (30 ,ug of total protein) and the co-factors required for the enzymes to metabolize BP-3,6-Q . In the experiments involving the use of oxygen-free radical scavengers, superoxide dismutase (SOD) and catalase were added into the reaction mixture. The reaction mixture (200 gl total volume) was incubated at 370C for 2 h in the dark. The optimum conditions required for the reductive metabolism of BP-3,6-Q catalysed by the enzymes were obtained from preliminary experiments. At the end of the incubation period, 25 jil of the reaction mixture was withdrawn and used to estimate the amount of the cofactors (NADH in the case of NQOl and NADPH in the case of P450 reductase) that remained unoxidized using the INT reagent (Nachlas et al, 1960). pUB3 DNA was isolated and purified from the remaining reaction mixture using phenol-chloroform extraction and ethanol precipita-NQO1 reduces quinone mutagenicity 711 vacuum, resuspended in Tris-EDTA buffer and used to transform competent ES87 cells. In similar experiments, BP-3,6-Q was replaced with 15 gM hydroquinone solubilized in ethanol. In related experiments, purified rat and human NQO1 enzymes were included with hydroquinone for adduction with pUB3; this was to study the role of NQOl in further metabolism of hydroquinone and its effect on hydroquinone-induced mutagenicity.
Transformation of competent ES87 cells and selection of the mutants Competent ES87 cells were prepared and transformed either with the control pUB3 DNA or that adducted with the BP metabolites, according to the published procedures (Rodriguez and Leochler, 1993;. SOS induced (140 J m-2) and uninduced ES87 cells were transformed with 1 jg of the pUB3 DNA by electroporation using the Gene Pulser (Bio-Rad, CA, USA) at 2.5 kV, 250 uF and 200 ohms according to the manufacturer's instructions. The transformants were allowed to recover in SOC medium for 1 h and plated on lactose minimal plates (LM plates) after diluting appropriately to select the mutants growing as large, blue colonies. The mutants were further screened by a second round of selection on LM plates followed by melibiose minimum (MM) plates, as described earlier . A small portion of the transformants was diluted and plated on LB plates containing ampicillin to determine the total number of transformants. Mutation frequency (MF) was calculated on the basis of the number of mutants growing on the MM plates and the total number of the ampicillin-resistant transformants growing on the LB plates. DNA was isolated from the mutant colonies growing on the MM plates and used to transform competent DH5-ox cells. Double-stranded DNA free of contaminating RNA and protein was isolated from the DH5-x cells and was analysed for mutations of the target supF tRNA gene by DNA sequencing. DNA sequencing was performed with T7 DNA polymerase using the sequenase kit version 2.0, according to the manufacturer's instructions. It may be noteworthy that every mutation was picked up from a single transformation to avoid the generation of siblings of individual mutations.
Competition between P450 reductase and NQ01 for BP-3,6-Q metabolism and mutation In order to assess the capacity of P450 reductase and NQO1 to compete for the reductive metabolism of BP-3,6-Q and to result in mutation, the microsomes possessing P450 reductase and the cytosol possessing NQO1 activity from the COS 1 cells transfected separately with the corresponding cDNAs were used. The enzyme samples were mixed in different proportions and used in the incubation mixture containing BP-3,6-Q, pUB3 DNA and the cofactors required for the reductive metabolism of BP-3,6-Q. In different experiments, either the amount of P450 reductase or that of NQO1 was changed by keeping that of the other one constant. At the end of the incubation period, DNA was isolated and used to transform competent, SOS-induced ES87 cells, and the mutation frequency was determined as described above.

RESULTS
Activities of microsomal P450 reductase and NQ01 in untransfected and transfected monkey kidney COS1 cells The microsomal P450 reductase and cytosolic NQO1 activities in the untransfected and transfected monkey kidney COS1 cells are shown in Table 1. The untransfected COS1 cells possessed an undetectable amount of endogenous NQO1 and a very low amount of P450 reductase activity (Table 1; Joseph and Jaiswal, 1994). The pMT2-P450 reductase-transfected COS1 cells produced 57fold higher P450 reductase activity than the untransfected control cells. Similarly, the pMT2-NQO1-transfected COS1 cells expressed 1192-fold higher NQO1 activity than the untransfected COS1 cells. These higher activities of P450 reductase and NQO1 allowed us to use the corresponding subcellular fractions (microsomes for P450 reductase and cytosol for NQO1) for metabolic reduction of BP-3,6-Q to study mutagenicity of quinones and the role of these enzymes in quinone mutagenicity.

Mutation frequency
In general, mutation frequency, either spontaneous or that due to the chemical modification of the pUB3 DNA, was higher (approximately two-to threefold) in the SOS-induced cells than in those that were not subjected to SOS induction (data not shown). With the exception of the quantitative difference in mutation frequency, no other change in response to the SOS induction of the ES87 cells was noticed. Therefore, only those results obtained with the SOSinduced ES87 cells are presented hereafter. In addition, the incubation of pUB3 DNA with BP-3,6-Q showed similar frequency and type of mutations as those observed with pUB3 + BP-3,6-Q mixed and incubated with untransfected COS 1 cell microsomes or cytosol
The chemical modification of the pUB3 DNA with either BPDE or BP-3,6-Q resulted in significant increases in the mutation frequency compared with that of the spontaneous mutations with the control (untreated) pUB3 DNA (Fig IA). The treatment of pUB3 DNA with BPDE and BP-3,6-Q resulted in a sevenfold and a fourfold increase, respectively, in the mutation frequency, compared with the background mutation frequency. The metabolic reductive activation of BP-3,6-Q by P450 reductase (one-electronreducing enzyme) further increased the mutation frequency of BP-3,6-Q by approximately twofold, compared with the unmetabolized BP-3,6-Q, and by 8.2-fold, compared with the frequency of spontaneous mutations (Figure iB). Interestingly, the pUB3 mutations induced by P450 reductase-activated BP-3,6-Q were eliminated/ prevented by superoxide dismutase (SOD) and catalase ( Figure  1B). Thirty units of SOD alone was less effective than 40 units of catalase and a combination of 7.5 units of SOD with 10 units of catalase ( Figure 1B). The P450 reductase-activated BP-3,6-Qinduced mutations in pUB3 were also significantly reduced as a result of the inclusion of NQO1 with P450 reductase in the metabolism of BP-3,6-Q (Figure 2A). Increasing the concentration of NQOl with a constant concentration of P450 reductase showed a NQOI concentration-dependent decrease in pUB3 mutations caused by BP-3,6-Q (Figure 2A). In a similar experiment, increasing the concentration of P450 reductase with a constant concentration of NQOI increased the pUB3 mutations caused by BP-3,6-Q ( Figure 2B). The slow increase in mutation frequency with increasing concentration of P450 reductase may be due to the presence of large quantities of NQO1, which is known to compete with P450 reductase and to detoxify quinones ( Figure 2B). In another related experiment, the increase in the concentration of NQOI caused steady but less significant decreases in BP-3,6-Q (unmetabolized)-induced pUB3 mutations ( Figure 2C). It may be noteworthy that incubation of BPDE with P450 reductase and of NQO1 individually and in combination had no effect on the mutation frequency, compared with BPDE alone (data not shown).
British Journal of Cancer (1998) 77(5) Figure 2 Competition between cytochrome P450 reductase and NQ01 to metabolize benzo(a)pyrene-3,6-quinone and its role in quinone mutagenicity. (A) pUB3 DNA was incubated with DMSO or BP-3,6-Q, a constant amount (30 jg of protein) of COS1 cell microsomes expressing high levels of P450 reductase and varying amounts (jg of protein) of COS1 cytosolic extract expressing very high levels of NQ01. (B) pUB3 DNA was incubated with DMSO or BP-3,6-Q, a constant amount (jg of protein) of COS1 cytosolic extract expressing very high levels of NQ01 and varying amounts (igg of protein) of COS1 cell microsomes expressing high levels of P450 reductase. (C) pUB3 DNA was incubated with DMSO or BP-3,6-Q and varying amounts (gg of protein) of COS1 cytosolic extract expressing very high levels of NQ01. In each case, after the incubation, the pUB3 DNA was isolated from the reaction mixture and was used to transform SOS-induced ES87 cells. The mutation frequency was determined as described in the text. The data represent the mean ± s.e. of three independent experiments Mutation spectra The mutation frequencies of the various treatments of pUB3 DNA are shown in Table 2. The location of mutations by the various agents used in the present study are shown in Figure 3. It is noteworthy that only a portion of the total mutations from Table 2, which includes all types of mutations in proper ratios, are shown in Figure 3. The analysis of mutation frequencies and the mutation spectra of the different treatments showed that they were distinctly different, suggesting the chemical specificity of mutations (Table 2 and Figure 3). Among spontaneous mutations (pUB3 alone), the single most abundant mutation was the deletion of 51 bp in the region extending from bp 68 to bp 119 of the supF tRNA gene. BPDE exhibited a very high specificity for the G:C -* T:A transversion, which accounted for 51% of all the mutations. It was found that the mutational frequency of BPDE-induced G:C -* T:A transversions was 46-fold higher than spontaneous mutations. Fifty per cent of BPDEinduced G:C -4 T:A transversions was associated with the base G at nucleotide position 15 of the supF tRNA gene of the pUB3 ( Figure  3). Frameshift mutations were very rare in the case of BPDE.
nucleotides 172 and 176 at a mutation frequency of 0.60 (Table 2). This deletion of a single cytosine from the DNA sequence of 5'-CCCCC-3' was not observed in spontaneous mutations or mutations caused by BPDE, unmetabolized BP-3,6-Q or P450 reductase-activated metabolic products of BP-3,6-Q (semiquinone and ROS). The results from previous studies and those from the present report were used to generate a model to understand the role of various enzymes in the metabolic activation, detoxification, mutagenicity and possibly carcinogenicity of BP-3,6-Q (Figure 4).

Hydroquinone mutagenicity
The mutational spectra of BP-3,6-HQ (generated as a result of the two-electron reduction of BP-3,6-Q catalysed by NQO1) was very interesting, as described above. It caused specific deletion of a single cytosine from the sequence 5'-CCCCC-3' in the supF tRNA observed in spontaneous mutations and the mutations caused by BP-3,6-Q and P450 reductase-catalysed metabolites of BP-3,6-Q (BP-3,6-SQ and ROS) ( Table 3). These observations raise three very important questions. First, do other hydroquinones also cause similar mutations involving deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' as observed with BP-3,6-HQ? Second, does BP-3,6-HQ undergo further metabolism catalysed by NQO1 to generate unidentified metabolites that cause deletion mutation of a single cytosine? Third, does BP-3,6-HQ undergo auto-oxidation by atmospheric oxygen to generate ROS that causes specific deletion of a single cytosine from the sequence 5'-CCCCC-3'? Several mutagenesis experiments were performed with BP-3,6-HQ and a second hydroquinone, benzoquinone hydroquinone (HQ), to address these questions (Table 3). HQ, such as BP-3,6-HQ, caused specific deletion of a single cytosine from the sequence 5'-CCCCC-3' of the supF tRNA gene.
gene at a high frequency. This type of deletion mutation was not However, the mutation frequency of cytosine deletion as a result     Table 2, which included all types of mutations in proper ratios, are shown. The numbering used is similar to that reported earlier (16); the promoter is between bp 24 and 58, the pre-tRNA is between bp 59 and 98 and the tRNA gene is between bp 99 and 183. The base undergoing mutation is underlined. A denotes deletion. The mutational spectra of pUB3 + BP-3,6-Q in the absence and presence of COS1 cell microsomes expressing endogenous (low) activity of P450 reductase and COS1 cell cytosol expressing undetectable levels of NQ01 were the same, therefore data are shown only for pUB3 + BP-3,6-Q  Values represent mean ± s.e. of three independent experiments. aMutational spectra other than deletion of base C in all the cases were more or less similar to the spontaneous mutations as shown in Figure 3 and Table 2. bCOS1 cells were transfected with cDNA encoding NADPH:cytochrome P450 reductase. The transfected cells expressed a 57-fold higher amount of P450 reductase activity, compared with untransfected COS1 cells as shown in Table 1. cCOS1 cells transfected with NQO1 cDNA. The cytosolic fraction of transfected cells expressed 1192-fold higher levels of NQO1 activity, compared with the untransfected cells. d and eThe purified human and rat NQO1 were obtained from Dr D Ross, School of Pharmacy, Denver, Colorado, USA. Both human and rat NOO1 enzymes are known to catalyse high-affinity reduction of BP-3,6-Q to BP-3,6-HQ (6). of HQ was 50% lower than that of BP-3,6-HQ (Table 3). The results also demonstrate that further metabolism of BP-3,6-Q and HQ with large amounts of purified human and rat NQO1 did not show any significant increase in mutational frequency of deletion of a single cytosine from 5'-CCCCC-3' in the supF tRNA region of pUB3 DNA (Table 3). In addition, incubation of SOD and catalase with HQ failed to decrease the frequency of deletion of the cytosine (Table 3).

DISCUSSION
The multistep process of chemical carcinogenesis begins with the metabolic activation of the procarcinogens to the ultimate, genotoxic metabolite(s) that interact with the DNA and other macromolecules, resulting in DNA and membrane damage (Guengerich, 1992). The presence of the unrepaired DNA damage during DNA replication process results in cellular genetic changes influencing the expression of growth-regulatory genes, culminating in the genesis of cancer (Harris, 1991). As metabolism of the procarcinogens is the pivotal step determining the ultimate genotoxic and therefore the carcinogenic potential of the chemicals, selective detoxification of the procarcinogenic chemicals by modulating the activities of the detoxification enzymes has attracted much attention as a feasible chemopreventive mechanism (Talalay et al, 1988;Prochaska et al, 1992;Morse et al, 1993).
Quinones (e.g. benzo(a)pyrene quinones, benzoquinones and naphthoquinones) belong to a class of chemicals that are known to cause cytotoxicity (O'Brien, 1991). However, the mutagenicity and carcinogenicity of various quinones remain relatively unknown. P450 reductase and NQO1 are the two most important enzymes within the cells that catalyse activation and detoxification of quinones respectively . It may also be noteworthy that quinones are electrophilic compounds that can bind to the DNA without undergoing enzymatic metabolism (O'Brien, 1991). For example, a quinone metabolite of BP, the 7,8-quinone, has been shown to form covalent adducts via Michael addition with calf thymus DNA and the plasmid pGEM3 DNA without further metabolism of the parent compound (Shou et al, 1993). One-electron reductive activation of quinones by enzymes, such as P450 reductase, generates semiquinones and reactive oxygen species (ROS) that have a high affinity for binding to the DNA (O'Brien, 1991;Joseph et al, 1994;Talalay et al, 1995). On the other hand, two-electron reduction of quinones by NQO1 results in the formation of hydroquinones that are much more stable than semiquinones and are removed by glucuronidation and other conjugation reactions (O'Brien, 1991;Joseph et al., 1994;Lind, 1985;Talalay et al, 1995). Therefore, the NQO1 pathway of metabolism of quinones is considered protective compared with the P450 reductase pathway. Previously, we reported that P450 reductase-activated BP-3,6-Q (BP-3,6-SQ and ROS) binds specifically to the deoxy guanosine residues of the DNA, resulting in the formation of DNA adducts . We also reported that NQO1 competed with the P450 reductase and specifically prevented the binding of P450 reductase-activated BP-3,6-Q to the DNA .
In the present report, we demonstrated that binding of unmetabolized and enzymatically metabolized BP-3,6-Q to the DNA was mutagenic. The BP-3,6-Q-induced mutation frequency was 1.5fold lower than BPDE but fourfold higher than spontaneous mutations. P450 reductase activated BP-3,6-Q into products (BP-3,6-SQ and ROS) that further increased the mutation frequency to eightfold higher than the spontaneous mutations. Analysis of the mutation spectra revealed several characteristic features concerning the mutational specificity of BPDE, BP-3,6-Q and the reductive metabolites of BP-3,6-Q, including BP-3,6-SQ, ROS and BP-3,6-HQ. In general, the mutation spectra of BPDE was distinctly different from that of BP-3,6-Q and its reductive metabolites. While BPDE resulted almost exclusively in basepair substitution mutations, BP-3,6-Q and its reductive metabolites caused both frameshift mutations, in particular deletions and base substitutions. BPDE-induced G:C -+ T:A transversions and the identification of a mutational hot spot at GI 15 of the supF tRNA gene were in agreement with previous reports (Rodriguez and Leochler, 1993;Ruggeri et al, 1993).  Figure 4 Model for benzo(a)pyrene quinones mutagenicity and carcinogenicity. Three pathways of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) activation and detoxification are shown. First pathway involves direct binding of BP-3,6-Q to the DNA resulting in fourfold increase in mutagenicity of DNA, compared with spontaneous mutations. However, no specificity of mutations are observed as a result of the direct binding of BP-3,6-Q to the DNA. In the second pathway, the one-electron-reducing enzymes (e.g. P450 reductase) metabolically reduce the BP quinone (BP-3,6-Q) to semiquinone (BP-3,6-SQ), which enters in the redox cycling to generate reactive oxygen species (ROS). The generation of semiquinone and ROS causes a further increase in mutation frequency, resulting in specific mutations as shown. In the third pathway, the two-electron-reducing enzyme (e.g. NQO1) competes with P450 reductase and detoxifies BP-3,6-Q resulting in reduction/prevention of P450 reductase-activated BP-3,6-Q-induced mutations. However, in this pathway, conversion of BP-3,6-Q to hydroquinone (BP-3,6-HQ) by NOO1 also led to specific frameshift mutations involving deletion of a single cytosine from the sequence 5'-CCCCC-3'. This type of mutation was not detected with BP-3,6-Q and P450 reductase-activated BP-3,6-Q (BP-3,6-SQ and ROS). Mutation frequencies ( Considerable differences in the mutational specificity were also observed among unmetabolized BP-3,6-Q and its reductive metabolites, suggesting that the actual mutagenic species in each case may be different. In spite of its capacity to cause frameshift mutations, the unmetabolized BP-3,6-Q did not show very high specificity for any particular kind of mutation, compared with the spontaneous mutations. Unlike the parent compound, the reductive metabolites of BP-3,6-Q showed specific affinity for siteand base-specific mutations. One-electron reductive activation of BP-3,6-Q by P450 reductase into products (BP-3,6-SQ and ROS) resulted in a very high affinity for mutations involving the base G. This was expected based on the earlier observations (Shou et al, 1993;Joseph and Jaiswal, 1994) that the quinones exhibit very high affinity for binding with the deoxy guanosine base of DNA. The predominant base deletion was that of the base G. The frequent base substitution mutations observed with the BP-3,6-Q metabolized by P450 reductase were G:C -> A:T, G:C -4 C:G; G:C -> T:A; A:T -* G:C and A:T -> T:A. Inhibition of the increased mutagenicity of P450 reductase-activated BP-3,6-Q (BP-3,6-SQ and ROS) by the scavengers of ROS indicated that ROS generated during the metabolic activation of BP-3,6-Q by P450 reductase was actually responsible for the increased mutagenicity of BP-3,6-Q. This observation with BP-3,6-Q is also supported by recent studies on mutagenicity of naphthoquinones and benzoquinones using Salmonella strains sensitive to oxidative mutagens (Hakura et al, 1994(Hakura et al, , 1995. The quinones, in general, enter into redox cycling to produce semiquinones and ROS (Figure 4). The ROS generated in the redox cycling causes oxidative stress and DNA damage, including mutagenicity and possibly carcinogenicity (Figure 4).
The failure of P450 reductase to enhance the mutagenicity of BP-3,6-Q in the presence of an excess amount of NQO1 indicated that NQO1 competed with P450 reductase for the metabolic detoxification of the quinone, leading to the prevention of onset of redox cycling and generation of mutagenic semiquinones and ROS. The products (hydroquinones) of the two-electron reduction of BP-3,6-Q catalysed by NQOl are quite stable in contrast to those of the semiquinones generated by the P450 reductase and are detoxified by glucuronidation, sulphation and/or glutathione conjugation reactions ( Figure 4). Thus, these results further confirm earlier observations Talalay et al, 1995) suggesting that NQOI functions as a cellular control device preventing the generation of semiquinones and the associated oxidative stress so as to prevent the genotoxicity and possibly the carcinogenicity of BP-3,6-Q.
In summary, the results of the present study clearly indicate that one-electron-reducing enzymes (e.g. P450 reductase) activate BP-3,6-Q into metabolic products (semiquinones and ROS) that are mutagenic and possibly carcinogenic (Figure 4). This conclusion has two important implications. Firstly, the quinones are mutagenic compounds and, secondly, the mutagenicity of BP is not only caused by BPDE but also by BP-3,6-Q. The various results also support the proposed chemopreventive role for NQO1 (Lind et al, 1982;Chesis et al, 1984;O'Brien, 1991;Monks et al, 1992;Joseph and Jaiswal, 1994;Talalay et al, 1995) (Figure 4). However, a careful analysis of the mutation spectra induced by metabolites of BP-3,6-Q generated by NQOI suggests that further studies are required to attribute an exclusive chemoprevention role for NQO1. This is because BP-3,6-HQ, a product generated during the metabolism of BP-3,6-Q catalysed by NQO1, was not completely nongenotoxic. The hydroquinone (BP-3,6-HQ) exhibited a very high sequence-specific mutation, i.e. deletion of a single cytosine from the sequence of 5'-CCCCC-3'. Similar deletion mutations were also observed with another hydroquinone, i.e. benzoquinone hydroquinone (HQ). The deletion of a single cytosine from the sequence 5'-CCCCC-3', however, was not observed in spontaneous mutations and mutations with BP-3,6-Q and P450 reductaseactivated BP-3,6-Q (BP-3,6-SQ and ROS). This clearly suggests that hydroquinones specifically cause deletion of a single cytosine from the DNA sequence 5'-CCCCC-3'. In addition, the deletion of cytosine is expected to be due to direct binding of hydroquinones to the DNA because the frequency of mutations was unaffected upon incubation of HQ with purified NQO1 and SOD + catalase. Chemically induced base deletions and substitutions are known to cause activation of oncogenes and inactivation of tumoursuppressor genes, leading to tumour development (Balmain and Brown, 1988;Hollstein et al, 1991;Vogelstein and Kinzler, 1992). It is unclear, at this time, whether the hydroquinone-specific deletion of cytosine will be observed in mammalian cells and will survive the DNA repair process to play a role in oncogenesis. It is especially important to study this in conditions in which hydroquinones may accumulate in cells as a result of the reduced/lack of expression of conjugating enzymes required for detoxification and elimination of hydroquinones from the cells.


extfieldsofstudy: Medicine Biology

text:
A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

Calcium sensing receptors are part of a growing G protein-coupled receptor family, which includes metabotropic glutamate, γ-aminoisobutyric acid, and pheromone receptors. The distinctive structural features of this family include large extracellular domains that bind agonist and large intracellular, carboxyl-terminal domains of as yet undefined function(s). We have explored the contribution(s) of the carboxyl terminus of the human calcium sensing receptor (CaR) by assessing extracellular Ca2+-mediated changes in intracellular Ca2+ in individual HEK-293 cells transfected with CaR clones. In-frame fusion of EGFP to the carboxyl terminus of CaR had no effect on either the dose response for extracellular Ca2+ activation or CaR desensitization. Carboxyl-terminal truncations, fused in-frame with EGFP (CaRΔ1024-EGFP, CaRΔ908-EGFP, CaRΔ886-EGFP, and CaRΔ868-EGFP), were assessed for alterations in Ca2+-dependent activation or desensitization. Significant effects on the dose-response relation for extracellular Ca2+ were observed only for the CaRΔ868 truncation, which exhibited a decreased affinity for extracellular Ca2+ and a decrease in the apparent cooperativity for Ca2+-dependent activation. The alterations in extracellular Ca2+ affinity and cooperativity observed with CaRΔ868 were recapitulated by a point mutation, T876D, in the full-length CaR-EGFP background. All truncations with wild type dose-response relations exhibited desensitization time courses that were comparable to the full-length CaR, whereas the CaRΔ868 receptor desensitized completely after two exposures to 10 mmCa2+. Interestingly, the CaR point mutation T876D exhibited desensitization comparable to wild type CaR, suggesting that this mutation specifically modifies CaR cooperativity. In conclusion, these studies suggest that amino acid residues between 868 and 886 are critical to the apparent cooperativity of Ca2+-mediated activation of G proteins and to CaR desensitization.

Calcium sensing receptors (CaR) 1 have been identified in many tissues, including neurons (1,2), retinal epithelium (3), kidney (4), intestine (5,6), parathyroid (7), bone (8), keratinocytes (9), and fibroblasts (10). Its role in many of these tissues has yet to be fully defined, although a common functional consequence of extracellular Ca 2ϩ activation of CaR is a G qmediated increase in phospholipase C␤ activity leading to increases in phosphoinositide hydrolysis and a release of Ca 2ϩ from thapsigargin-sensitive intracellular stores (11). In some cell types, CaR activation also leads to rapid alterations in c-myc expression (5) or activation of the mitogen-activated protein kinase cascade (10), suggesting coupling to multiple signal transduction pathways in any given cell type/tissue is possible.
Calcium sensing receptors represent a novel member of a growing family of G protein-coupled receptors (family C), which include metabotropic glutamate receptors (mGluRs) (12,13), calcium sensing receptors (CaRs) (7,14), ␥-aminoisobutyric acid receptors (15), and a newly cloned multigene family of putative pheromone receptors identified in the rat vomeronasal organ (16 -18). Agonists for this family of receptors represent simple molecules present in the extracellular milieu, e.g. amino acids and ions, and these receptors most likely developed as a fusion between bacterial periplasmic binding proteins and the seven transmembrane/signaling domains common to G protein-coupled receptors (19,20). Agonist binding occurs within the large extracellular NH 2 -terminal domain (21), rather than in a pocket defined by the seven transmembrane helices as is characteristic of most G protein-coupled receptors (22,23). A cluster of CaR mutations identified in patients with hyper-and hypoparathyroidism, which affect CaR affinity for Ca 2ϩ , cluster within the extracellular domain (11), suggesting this as the location of the Ca 2ϩ binding site(s). Activation of CaR by extracellular Ca 2ϩ is highly cooperative (Hill coefficients for activation in the range of 2-4) both in vivo (24) and in vitro (25), and although the molecular mechanism is undefined, it has been suggested to result from multiple Ca 2ϩ binding sites (25,26).
A second distinctive feature of many members of this receptor superfamily is the large intracellular carboxyl-terminal domain. Several mGluR genes are alternatively spliced to produce receptors having variations within the carboxyl terminus (27). Studies on heterologously expressed mGluR1 (a, b, or c) or mGluR5 (a or b) variants suggest that carboxyl-terminal differences affect receptor-mediated signaling (28,29) as well as the level of agonist-independent activity (30). Further, the carboxyl-terminal domain of mGluR1 has been shown to affect the sensitivity of the receptor to agonist in chimeras having identical NH 2 -terminal agonist-binding domains (28). Finally, the large carboxyl terminus may be involved in interactions with proteins that assist in transport of expressed receptors to the plasma membrane, as has been shown for Homer-mediated trafficking of mGluRs (31). Calcium sensing receptors also have a large carboxyl terminus (Ͼ210 amino acids). Truncations within the carboxyl terminus have recently been shown to severely limit phosphatidylinositol hydrolysis when the residual carboxyl terminus was limited to Ͻ15 amino acids, and a region between amino acids 874 and 888 has been identified as crucial to normal signal transduction (32).
In this report, we explore the functional consequences of carboxyl-terminal truncations in greater detail, utilizing an assay with enhanced sensitivity and time resolution, applied to individual cells. In order to resolve subtle differences in receptor function, we have characterized the truncations with respect to both the dose-response relation for Ca 2ϩ -dependent activation and CaR desensitization, since desensitization of G protein-coupled receptors often involves phosphorylation and/or conformation-dependent exposure of sequestration motifs within the carboxyl-terminal domain (33,34). Finally, all studies were performed with CaR constructs fused to EGFP at the carboxyl terminus, to allow identification of cells expressing receptors. We find that carboxyl-terminal fusion with EGFP does not affect the functional properties of CaR, and provides a useful marker for assessment of absolute expression levels of CaR within the cell. Modest truncations of the carboxyl terminus (CaR⌬1024, CaR⌬908, and CaR⌬886) had no effect on membrane localization, Ca 2ϩ dose response, or rates of desensitization, whereas truncation CaR⌬868 both altered the dose-response relation for Ca 2ϩ and enhanced desensitization. Finally, a point mutation T876D in the full-length CaR-EGFP background recapitulated the effects observed with CaR⌬868 with respect to the Ca 2ϩ dose-response relation, suggesting that this residue is critically involved in determining the cooperativity of G protein activation.

EXPERIMENTAL PROCEDURES
Materials-The full-length human CaR clone (in pBluescript) was provided by Dr. Jim Garrett of NPS Pharmaceuticals (Salt Lake City, UT); pEGFP was obtained from CLONTECH. Restriction enzymes were obtained from Life Technologies Inc. and New England Biolabs.
Plasmid Construction-PCR reactions were performed with PfuI polymerase (Stratagene) using 94, 53, and 72°C as denaturing, annealing, and elongation temperatures, respectively. All constructs were confirmed by restriction digests and automated sequencing. Full-length human CaR was subcloned into pCDNA3.1 (Invitrogen) after HindIII-NotI digestion (pCCaR). In order to facilitate identification of transfected cells, CaR and EGFP were sometimes expressed behind different promoters in the same mammalian expression vector (described as CaR construct/EGFP). The EGFP gene was excised from its original vector (pEGFP-N1; CLONTECH) by NheI-NotI digestion and inserted downstream of the SV40 promoter of pCCaR after digestion with BstBI-AvrII (replacing the neomycin resistance gene). To create a CaR-EGFP fusion protein, a two-step cloning procedure was used. First, a PCR product was generated using a 5Ј primer which anneals before the XmaI site of pBluescript/CaR (TTC CGC AAC ACA CCC ATT GTC AAG G), and a 3Ј primer in which the CaR gene stop codon was replaced by a BclI site (GCT GAT CAA CTA CGT TTT CTG TAA CAG T). After PCR amplification, the product was digested with XmaI and subcloned back into the original template using XmaI and a T4 polymerase filled-in NotI site. The final chimeric gene was created by excising the mutated CaR gene from pBluescript/mutCaR using NotI-BclI and inserting it between the NotI-BamHI sites of pEGFP (CLONTECH). Two of the truncations were constructed by direct subcloning of portions of the CaR gene from pBluescript/CaR into pEGFP (CLONTECH). For CaR⌬886-EGFP, the CaR gene was cut and subcloned into pEGFP-N1 with HindIII-XmaI. The same was done for CaR⌬908-EGFP using HindIII-BamHI and pEGFP-N3. For two other truncations (CaR⌬1024-EGFP and CaR⌬868-EGFP), site-directed mutagenesis PCR products were generated using pBluescript/CaR as a template, a common 5Ј primer (GAA TTG TAA TAC GAC TCA CTA TAG GGC GA) and 3Ј primers containing an in-frame BamHI site (CGG TCG GAT CCA AGT CCG TTT CCC C for CaR⌬1024-EGFP and GGC GAT GGA TCC GGT GTT GCG GGA TGG C for CaR⌬868-EGFP). The PCR products were cut with HindIII-BamHI, purified by electrophoresis on 1% agarose gels, isolated, and subcloned into pEGFP. CaR⌬868 was also cloned into pCCaR/EGFP, replacing the full-length CaR gene. The PCR strategy described above was performed, using the same 5Ј primer and a 3Ј primer containing a stop codon immediately before a BamHI site (CAA CGC GGA TCC TAG ATG GTG TTG CGG GAT GG). CaR-T876D-EGFP was produced by PCR-based site-directed mutagenesis using the CaR-EGFP construct as template (5Ј primer, GCA CCT CCT CGA TGG TGT TGC G, 3Ј primer GTT GCA GCG ACG CAG CTC ACG C). Fig. 1 illustrates the topology of the human CaR, the location within the carboxyl-terminal domain of the various truncations, and the fusion proteins that were produced with EGFP.
Cell Culture/Transfections-HEK-293 cells were grown in high glucose Dulbecco's modified Eagle's medium (Life Technologies Inc.) supplemented with 10% bovine serum albumin (Sigma), 50 units/ml penicillin, and 50 g/ml streptomycin (37°C, 5% CO 2 ). Cells were transfected with 1 g of each construct using Superfect (Quiagen), plated on collagen-coated coverslips, and kept in a 5% CO 2 incubator at 37°C until use. Some constructs (CaR, CaR/EGFP, CaR-EGFP, CaR⌬868, and CaR⌬868-EGFP) were also stably transfected after linearization with SspI. The constructs CaR/EGFP and CaR⌬868/EGFP did not contain a neomycin resistance gene and were therefore cotransfected with linearized pCDNA3.1 in a 10:1 ratio. Cells were kept in medium containing 300 g/ml Geneticin (Life Technologies, Inc.). Colonies were selected after 3 weeks, and clones were confirmed by both cell fluorescence and CaR activity.
Confocal Microscopy-Coverslips containing transfected HEK-293 cells were fixed with acetone for 5 min at Ϫ20°C, mounted on glass slides with Vectashield (Vector Laboratories), and viewed with a Noran Oz confocal microscopy system. Selected regions of the field were excited at 340/380 nm (emission wavelength 510 nm) at 8-s intervals (8 frame averaging) on a Universal imaging system based on the MetaFluor software package. Background images at the same settings used during a particular experiment were obtained on regions of the coverslip devoid of cells. All solutions were osmolality-matched (290 -300 mOsm), measured on a Wescor 5500 vapor pressure osmometer (Salt Lake City, UT). Variations in extracellular Ca 2ϩ concentration were produced by isosmolar substitution for NaCl. All experiments were performed at room temperature (22-24°C). Multiple cells were analyzed from at least three independent transfections or cell passages (for stably transfected cell lines). Data were normalized/averaged as described in the text, and are presented as mean Ϯ standard error of the mean. Curves were fitted by least squares minimization using the Marquardt-Levenberg algorithm (NFIT, Island Products, Galveston, TX).

Comparison of the Functional Properties of Human CaR and
CaR-EGFP-To determine the localization and expression levels of the various CaR constructs, carboxyl-terminal fusions to EGFP were used (Fig. 1B). Preliminary studies were performed to confirm that EGFP did not affect the functional properties of Control experiments were performed in HEK-293 cells stably transfected with CaR-EGFP to determine whether EGFP interfered with measurements of intracellular Ca 2ϩ monitored by changes in fura-2 fluorescence. Cells expressing CaR-EGFP but not loaded with fura-2-AM were exposed to 0.5 and 10 mM extracellular Ca 2ϩ and the fluorescence measured at an emission wavelength of 510 nm (excitation wavelengths were 340 and 380 nm). The 340/380 fluorescence ratio was low (Ͻ0.5) and was unchanged by alterations in bath Ca 2ϩ . A second control determined the intracellular Ca 2ϩ responses to changes in extracellular Ca 2ϩ concentrations in the range from 1.5 to 30 mM in untransfected or vector (pCDNA3.1)-transfected HEK-293 cells. No significant changes in intracellular Ca 2ϩ were elicited in the absence of CaR.
A further means of comparing the functional properties of CaR and CaR-EGFP was to assess the rate of desensitization of the intracellular Ca 2ϩ response to repeated exposures to 10 mM extracellular Ca 2ϩ . Fig. 3 (A and B) illustrates the protocols used. For protocol 1 (illustrated in Fig. 3A), cells stably transfected with CaR were alternately exposed to 10 mM bath Ca 2ϩ for 2 min, followed by 0.5 mM Ca 2ϩ for 3 min. This protocol resulted in a modest rate of CaR desensitization over a 30-min period, as reflected by the peak response to successive 10 mM Ca 2ϩ applications. Protocol 2 (illustrated in Fig. 3B) utilized a 4-min exposure to 10 mM Ca 2ϩ , followed by a 2-min "recovery" period in 0.5 mM Ca 2ϩ . This protocol resulted in significant desensitization of CaR (Ϸ40 -50%) over a 30 min experiment. The responses to both protocols strongly suggest that the decline in response is due to CaR desensitization and not to slow depletion of intracellular Ca 2ϩ stores, since the extent of desensitization is proportional to the duration of receptor stimulation. Fig. 3C illustrates the time courses for desensitization of the peak intracellular Ca 2ϩ responses for CaR and CaR-EGFP for the two protocols, with all data normalized to the first peak response to 10 mM Ca 2ϩ of each cell. Carboxyl-terminal fusion of CaR to EGFP had no effect on the rate(s) of receptor desensitization.
Localization of CaR Carboxyl-terminal Truncations to the Plasma Membrane-Truncations of the carboxyl terminus of CaR were produced as fusion proteins with EGFP, as illustrated in Fig. 1B. Each construct was assessed for its cellular distribution prior to functional studies. Illustrated in Fig. 4 Table I. relation between EGFP fluorescence intensity (fluorescein isothiocyanate filter) and plasma membrane localization assessed by increases in intracellular Ca 2ϩ (fura-2 fluorescence changes) existed for all CaR-EGFP constructs.
Dose-Response Relations for Ca 2ϩ -mediated Activation of CaR Carboxyl-terminal Truncations-Experiments such as those illustrated in Fig. 2A were performed to assess the doseresponse relations for extracellular Ca 2ϩ -mediated activation of the CaR carboxyl-terminal truncations. Fig. 5 illustrates the results of fits to data averaged from at least three independent transfections for each construct, and Table I indicates the parameters of the fits. Only CaR⌬868-EGFP exhibited a difference in response to extracellular Ca 2ϩ , with both a decreased affinity for Ca 2ϩ (EC 50 of 10.9 mM) and a reduction in the Hill coefficient (from the range of 2.0 -2.9 down to 1.1). Since the ⌬868 truncation leaves a cytoplasmic tail of only 8 amino acids, we also determined the dose-response relation for an additional construct, CaR⌬868 (coexpressed with soluble EGFP) to eliminate the possibility that EGFP presented a physical barrier for access of G proteins to their sites of interaction with CaR. HEK-293 cell lines stably expressing CaR⌬868 were produced and the dose-response relationship for extracellular Ca 2ϩ determined. Results were similar for both the CaR⌬868-EGFP and CaR⌬868 constructs (data combined in Fig. 5 and Table I), implying that the decreased responsiveness to Ca 2ϩ is a result of the truncation and not of the carboxyl-terminal fusion to EGFP.
Desensitization of CaR Carboxyl-terminal Truncations-The progressive desensitization of peak intracellular Ca 2ϩ in response to repeated exposures to 10 mM extracellular Ca 2ϩ was assessed for all of the CaR carboxyl-terminal truncations, according to the two protocols described, i.e. protocol 1, 2 min 10 mM Ca 2ϩ /3 min 0.5 mM Ca 2ϩ ; and protocol 2, 4 min 10 mM Ca 2ϩ /2 min 0.5 mM Ca 2ϩ . Illustrated in Fig. 6 are the combined data for all CaR carboxyl-terminal truncations. For CaR⌬1024-EGFP, CaR⌬908-EGFP, and CaR⌬886-EGFP, desensitization was unchanged from that observed for full-length CaR (lines drawn in the figure represent desensitization of full-length CaR in response to the two protocols, redrawn from Fig. 3C). Desensitization of CaR⌬868-EGFP, however, was significantly enhanced, with full desensitization occurring after two applications of agonist.
Comparison of CaR⌬868 with a Point Mutation, CaR-T876D-EGFP-Results with the truncations suggest that the region between amino acids 868 and 886 is critical for normal functioning of CaR by several criteria; 1) CaR⌬868 exhibits a decrease in the apparent affinity for extracellular Ca 2ϩ , 2) the degree of cooperativity as reflected in the Hill coefficient is decreased, and 3) desensitization of CaR⌬868 is significantly increased. The region between amino acids 874 and 888 has been previously identified as important in normal signal transduction (32). We sought to identify residues(s) critical to the observed alterations in Ca 2ϩ signaling, and have identified a point mutation, T876D (produced in the background of fulllength CaR-EGFP), which recapitulates a specific property observed with the truncation, CaR⌬868-EGFP, i.e. T876D exhibits a reduced affinity for Ca 2ϩ with respect to activation, and a reduction in the apparent cooperativity for Ca 2ϩ -dependent activation as reflected in the Hill coefficient. Fig. 7A illustrates the dose-response relations. Parameters of the fits are listed in Table I.
CaR⌬868-EGFP and T876D are different, however, with respect to desensitization. Fig. 7B compares the second response to 10 mM Ca 2ϩ (after a 3-min recovery in 0.5 mM extracellular Ca 2ϩ ) for three CaR constructs, full-length CaR, CaR⌬868-EGFP, and T876D. It is clear that T876D exhibits only a modest degree of desensitization, comparable to that of the full-length wild type CaR, whereas CaR⌬868-EGFP exhibits strong desensitization. DISCUSSION The series of carboxyl-terminal truncations in this study contributes to a mapping of functional domains within the carboxyl terminus of CaR. Significant truncations, i.e. removal of up to 192 of the 218 amino acids within the carboxyl terminus, had no effect on either extracellular Ca 2ϩ -dependent activation of the G q -mediated signaling pathway or desensitization of the response upon repeated exposures to 10 mM extracellular Ca 2ϩ . This implies that amino acid residues 886 -1078 are not critical to these particular acute responses. A recent study utilizing calcium-stimulated phosphoinositide hy- drolysis as a measure of CaR activity demonstrated that truncations at 865 or 874 were inactive despite adequate cell surface expression (32). Therefore, the most significant and unexpected result in the present study, most likely attributable to the increased sensitivity and time resolution obtained when utilizing intracellular Ca 2ϩ as a measure of CaR activity, was that the CaR⌬868-EGFP truncation was active. CaR⌬868-EGFP activity was, however, distinctly different from the other truncations or the full-length CaR, exhibiting both a decrease in the apparent affinity for extracellular Ca 2ϩ -mediated G q activation and a decrease in the apparent cooperativity. This result was recapitulated by a point mutation, T876D, suggesting that this residue within the proximal carboxyl-terminal domain is pivotal in determining the affinity and apparent cooperativity for Ca 2ϩ activation of CaR. Further support for this conclusion comes from additional CaR clones having point mutations at neighboring residues: R873A, C874A, or S875A; all of these mutated CaRs had wild type properties with respect to Ca 2ϩ -dependent activation (data not shown). Further explo-ration of the region between residues 868 and 886 by sitedirected mutagenesis is under way.
A steep Ca 2ϩ -response relation is a hallmark of CaR activation, observed both in freshly isolated bovine parathyroid cells (24) and in cell lines expressing recombinant CaR (25,26). Highly cooperative activation is unique to CaR, and is not observed even in the related receptor family, mGluR (23,35,36). There is little precedent, therefore, for the molecular origins of CaR cooperativity. Several explanations are possible, as initially suggested by Ruat et al. (26): 1) multiple Ca 2ϩ binding sites within the extracellular domain; 2) cooperative interactions between multiple CaR; or 3) highly cooperative, efficient activation of G proteins. There is as yet no information about the existence of multiple binding sites for Ca 2ϩ on CaR, since high affinity, selective agonists and/or antagonists suitable for binding studies have not yet been identified. A recent report supports the interaction among multiple CaR as a possible source of cooperativity, since CaR has been shown to be present as covalent disulfide-linked dimers in membranes (37). Further studies are needed, however, before the functional consequences of CaR dimerization are understood. The present results suggest that the third possibility, i.e. highly cooperative, efficient activation of G proteins, may be the explanation for the apparent cooperativity of Ca 2ϩ activation of CaR. A single  Fig. 2A were performed on HEK-293 cells transiently transfected with the CaR carboxyl-terminal truncations illustrated in Fig. 1B. Data from at least 40 cells from two to three independent transfections were averaged at each concentration. All data were fitted with the Hill equation, and the fit parameters are listed in Table I. Symbols are: CaR (closed circles), CaR-EGFP (closed triangles), CaR⌬1024-EGFP (closed squares), CaR⌬908-EGFP (closed diamonds), CaR⌬886-EGFP (closed inverted triangle), and CaR⌬868-EGFP (closed hexagons).

TABLE I Comparison of dose-response relations obtained for full-length human
CaR and carboxyl-terminal truncations, expressed in human embryonic kidney cells Data from at least 50 cells from at least three independent experiments (CaR and CaR-EGFP, CaR⌬868) or three independent transfections (CaR⌬1024-EGFP, CaR⌬908-EGFP, CaR⌬886-EGFP, and CaR⌬868-EGFP) were combined, normalized to the peak response at 30 mM Ca 2ϩ , and fitted with the following equation  (25,26). One possible explanation of variable cooperativity is that the various agonists differ in their ability to induce the highly cooperative conformation of the carboxyl-terminal domain. There is precedence for a differential ability of full and partial agonists to induce active conformations of G proteincoupled receptors (38 -40).
The amino acid residues between 868 and 886 contain no putative phosphorylation sites. In addition, there is no sequence homology with other G protein-coupled receptors, except a dipeptide, ST, which aligns with the same two residues within the carboxyl terminus of the two mGluR5 splice variants, mGluR5a and b. The threonine residue in mGluR5a is a protein kinase C phosphorylation site (41) and is responsible for the intracellular Ca 2ϩ oscillations observed upon activation of these receptors (41,42). The comparable amino acid residue in mGluR1, which does not exhibit intracellular Ca 2ϩ oscillations, is aspartic acid (41). This region, and in particular, these residues, are therefore implicated as part of the G protein interaction site (in combination with the i2 intracellular loop). We therefore mutated the threonine residue in CaR to aspartic acid. Although intracellular Ca 2ϩ oscillations were still observed in CaR-T876D (data not shown), the cooperativity with respect to activation was altered, strongly implicating this amino acid residue as critical to the G protein interaction site, specifically mediating highly cooperative G protein activation.
A second feature of CaR function, which is localized to the region of the proximal carboxyl terminus between amino acids 868 and 886, is acute desensitization of the peak CaR-mediated response. The CaR⌬868-EGFP truncation desensitizes very rapidly compared with less severe truncations or wild type CaR. All of the carboxyl-terminal domain's putative protein kinase A and C sites (43) are eliminated in the CaR⌬886-EGFP truncation, which desensitizes normally. Therefore, these protein kinase sites do not participate in acute desensitization of CaR. Additional potential protein kinase C sites are localized to intracellular loops; in particular, there is one in the i3 loop that contributes, in many G protein-coupled receptors, to G protein activation. CaR⌬886-EGFP may desensitize normally because the amino acid residues between 868 and 886 retard access of the proteins involved in desensitization to the relevant phosphorylation site(s). Direct interactions between G protein-coupled receptor kinases and possibly arrestin with muscarinic receptors have been functionally demonstrated (44), although the consensus binding site(s) on the receptor have not been identified. Deletion of the amino acid residues of CaR between 868 and 886 may expose potential regulatory protein interaction site(s), leading to more rapid and complete phosphorylation and desensitization of CaR. Further studies with sitedirected mutants that eliminate all potential phosphorylation sites on CaR will be required to confirm this possibility.
Although the distal carboxyl terminus (from amino acids 886 -1078) is not involved in modulating acute CaR activation (reflected in the dose-response relation and desensitization rate), there are other potential role(s) for this domain. Several mGluR1 genes are expressed as slice variants, which differ primarily in the length of the carboxyl terminus (27). The longer splice variant of mGluR1 exhibits significant agonistindependent activity (30), and the carboxyl-terminal domain of  Table I. B, comparison of second response to application of 10 mM Ca 2ϩ for three constructs: full-length CaR, CaR⌬868-EGFP, and T876D, as a percentage of the first response. Experiments were performed as in Fig. 3A, i.e. there was a 3-min interval in 0.5 mM Ca 2ϩ between the first and second 2-min applications of 10 mM Ca 2ϩ . this variant (plus the i2 loop) confers enhanced sensitivity to agonist with respect to G protein activation (28). Although only a single CaR gene has been studied to date, functional differences have been observed among CaR cloned from different species (45), and several additional partial clones that localize to different mouse chromosomes have been identified (14). A family of CaR genes may therefore soon be available for more detailed sequence/function comparisons within the carboxyl terminus as well as throughout the protein.
Another potential role for the distal carboxyl terminus of CaR may be in trafficking and/or targeting of the receptor within particular cell membrane domains. CaR is highly expressed in various regions of the brain (46), and a subcellular localization within pre-and/or post-synaptic domains (1,47), in analogy with mGluRs, may require interaction with targeting proteins such as Homer (31), which has been recently shown to participate in mGluR transport to the plasma membrane in an activity-dependent manner. Significant concentrations of both mGluRs and CaR have been observed within intracellular membranes in vivo (31,47), and thus a similar mechanism may operate with respect to CaR targeting. Finally, CaR is highly expressed in epithelia within the kidney (48) and intestine (6), and therefore the potential for interactions with the cytoskeleton or other proteins within the target membrane to anchor and/or retain CaR may occur via this domain. The available of a series of carboxyl-terminal truncations of CaR which retain activity but have variable deletions of potential protein-protein interaction domains will make hunting for regulatory protein partners straightforward.
In this study, we have begun to dissect the molecular roles for the carboxyl terminus of CaR. A proximal portion, within 20 amino acid residues of the membrane, contributes specifically to regulation of the cooperativity of G protein activation, as well as to acute receptor desensitization. The availability of functional EGFP-tagged receptor mutants and truncations makes further exploration of the role(s) for the carboxyl-terminal intracellular domain of CaR feasible, and may generate paradigms generalizable to the superfamily of G protein-coupled receptors including CaR, mGluRs, ␥-aminoisobutyric acid receptors, and pheromone receptors.


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Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits*

Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl 2 , purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays ; 40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes nucleosomes and stored as described previously (18). Core histones used in histone acetyltransferase assays were concentrated to ; 1.0 mg/ml and dialyzed into 15 m M Tris-HCl, 0.25 m M EDTA, 0.05% Tween 20, 2.5 m M dithiothreitol, 5.0% glycerol, pH 7.8 (DB) buffer. Saturated 12-mer nucleosomal arrays (12 octamers/DNA) were assembled by combining the 208–12 template with core histone octamers and performing the salt dialysis method previously as described (18). Reconstitutes were then concentrated to ; 1.0 mg/ml and dialyzed into DB buffer. Nucleosomal core particles were isolated by digestion of native chicken oligo- nucleosomes with micrococcal nuclease as described (19). Core particles were concentrated to ; 1.0 mg/ml and dialyzed in DB buffer. All samples were concentrated using an Amicon Centricon concentrator (molecular weight cutoff 10,000). Expression and Purification of Recombinant GCN5— A 50-ml culture of Escherichia coli BL21 cells containing pRSET-yGCN5 was grown in LB broth containing 0.1 mg/ml ampicillin to an A 600 ’ 0.30–0.50. Cells were then induced with 0.4 m M isopropyl-1-thio- b - D -galactopyranoside for 3 h. Recombinant Gcn5p was purified using a Ni 2 1 -agarose affinity column as described (7), concentrated to ; 70 m g/ml, and dialyzed into DB buffer. The purity of the isolated rGcn5p was determined by silver staining of overloaded SDS-polyacrylamide gels. Analytical Ultracentrifugation— Sedimentation velocity experiments were performed using a Beckman XL-A analytical ultracentrifuge equipped with absorption optics as described (20). The A 260 of the samples was between 0.6 and 0.8. The integral distribution of sedimen- tation coefficients was determined by the method of van Holde

Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions
have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl 2 , purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays ϳ40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.
Histone acetylation is a reversible dynamic process that occurs at specific lysine residues in the N termini of all the core histone proteins and has been correlated with several key biological processes, including nucleosome assembly and modulation of gene expression (1)(2)(3)(4). The recent discoveries of the specific histone acetyltransferases (HATs), 1 Hat1p (5, 6) and Gcn5p (7), have directly linked histone acetylation with nucleosome assembly and transcriptional activation, respectively. Hat1p and Gcn5p are specific examples of the two general families of HATs, termed HAT-A and HAT-B. The HAT-A enzymes reside in the nucleus and acetylate the core histone N termini following their assembly into nucleosomes and chromatin. HAT-B type enzymes are primarily cytoplasmic and acetylate only free histones prior to nucleosome assembly (6), although Hat1p has recently been localized in the nucleus as well (8).
Hat1p is the catalytic subunit of both the major cytoplasmic HAT-B complex and the nuclear HAT-A3 complex (8). Consistent with their roles in nucleosome assembly, recombinant Hat1p, the HAT-B complex, and the HAT A-3 complex all acetylate free histone H4 in vitro but do not acetylate H4 after its incorporation into nucleosomes (5,6,8,9). However, seemingly at odds with its role as a HAT-A enzyme, Gcn5p has been reported to acetylate free histones H3 and H4 but not nucleosomal H3 or H4 (10 -13). This result has been reconciled by a model in which Gcn5p acetylates nucleosomal substrates only when a component of specific multiprotein complexes and is supported by the recent identification of several Gcn5-Ada complexes that are capable of acetylating histone H3 and to a lesser extent H4 in nucleosomes (12, 14 -16). Nevertheless, because all previous studies of rGcn5p activity were performed using a very limited range of buffer and substrate conditions, it remains to be determined whether other proteins are absolutely required for the HAT activity of Gcn5p or whether these proteins coincidentally accentuate Gcn5p activity in vitro under the conditions tested previously.
To address this issue, we have assayed the HAT activity of rGcn5p over a wide range of NaCl or MgCl 2 concentrations, using an excess of free core histones, nucleosome core particles, and differentially folded nucleosomal arrays as substrates. Under optimal salt conditions, we find that both nucleosome core particles and nucleosomal arrays are efficiently acetylated by rGcn5p. Moreover, relative to core particles, highly condensed nucleosomal arrays are acetylated much more rapidly. Finally, whereas free histone H3 is polyacetylated by rGcn5p, nucleosomal H3 is only monoacetylated. These results demonstrate that when studied under appropriate solution conditions, rGcn5p both exhibits pronounced catalytic activity in the absence of other protein subunits and prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the H3 N termini changes upon assembly of free histones into nucleosomes and nucleosomal arrays.

EXPERIMENTAL PROCEDURES
Materials-Whole chicken blood was purchased from Pel-Freeze. BL21 cells containing plasmid pRSET-yGCN5 were obtained from Dr. David Allis (University of Virginia). DNA templates consisting of 12 tandem repeats of a 208-base pair sequence derived from the Lytechinus 5 S rRNA gene (208 -12 DNA) were grown and purified from plasmid using pPOL208 -12 as described previously (17). All chemicals were of reagent grade.
Assembly of 208 -12 Nucleosomal Arrays and Purification of Core Particles-Core histones were purified from chicken erythrocyte oligo-nucleosomes and stored as described previously (18). Core histones used in histone acetyltransferase assays were concentrated to ϳ1.0 mg/ml and dialyzed into 15 mM Tris-HCl, 0.25 mM EDTA, 0.05% Tween 20, 2.5 mM dithiothreitol, 5.0% glycerol, pH 7.8 (DB) buffer. Saturated 12-mer nucleosomal arrays (12 octamers/DNA) were assembled by combining the 208 -12 template with core histone octamers and performing the salt dialysis method previously as described (18). Reconstitutes were then concentrated to ϳ1.0 mg/ml and dialyzed into DB buffer. Nucleosomal core particles were isolated by digestion of native chicken oligonucleosomes with micrococcal nuclease as described (19). Core particles were concentrated to ϳ1.0 mg/ml and dialyzed in DB buffer. All samples were concentrated using an Amicon Centricon concentrator (molecular weight cutoff 10,000).
Expression and Purification of Recombinant GCN5-A 50-ml culture of Escherichia coli BL21 cells containing pRSET-yGCN5 was grown in LB broth containing 0.1 mg/ml ampicillin to an A 600 Ϸ0.30 -0.50. Cells were then induced with 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h. Recombinant Gcn5p was purified using a Ni 2ϩ -agarose affinity column as described (7), concentrated to ϳ70 g/ml, and dialyzed into DB buffer. The purity of the isolated rGcn5p was determined by silver staining of overloaded SDS-polyacrylamide gels.
Analytical Ultracentrifugation-Sedimentation velocity experiments were performed using a Beckman XL-A analytical ultracentrifuge equipped with absorption optics as described (20). The A 260 of the samples was between 0.6 and 0.8. The integral distribution of sedimentation coefficients was determined by the method of van Holde and Weischet (21) using Ultrascan data analysis software version 2.95 (Borries Demeler; University of Texas Health Science Center).
HAT Activity Assays-HAT activity assays were performed with 15 g of either free histone proteins, nucleosome core particles, or 12-mer nucleosomal arrays as substrates using a slightly modified version of the procedure described previously (22). In a final reaction volume of 60 l, 5 l (0.35 g) of rGcn5p was mixed with 0.  (23) or acetic acid/urea/Triton (AUT) (24) gel for gel analyses. Microsequencing Analysis-The N termini of histone H3 were sequenced with a Perkin-Elmer Applied Biosystems 477A gas/liquid protein sequencer using the N-terminal Edman degradation procedure. Briefly, free core histones were incubated with rGcn5p, 2.5 Ci of [ 3 H]acetyl-CoA, and 2.25 nmol of unlabeled acetyl-CoA for 90 min and then resolved on an 18% SDS-polyacrylamide gel. The histones were transferred to P SQ -polyvinylidene difluoride membrane (Millipore) at 1.5 mA/cm 2 in a Millipore semi-dry electrophoresis unit. The band corresponding to histone H3 subsequently was excised and sequenced as described.

Recombinant Gcn5p Acetylates Nucleosomes and Nucleosomal Arrays in the Presence of Optimal Concentrations of Monovalent or Divalent
Cations-Previous assays of rGcn5p activity have been performed exclusively in buffer containing 50 mM Tris-HCl and either no salt (7,10,13,25) or a mixture of 50 mM KCl plus 50 mM NaCl (12). To determine whether rGcn5p possesses the ability to acetylate nucleosomal substrates when studied over a wider range of salt conditions, HAT assays were conducted in the presence of 15 mM Tris-HCl and either 0 -300 mM NaCl or 0 -10 mM MgCl 2 , using free core histones, nucleosome core particles, and defined 12-mer nucleosomal arrays as substrates. The ability of rGcn5p to acetylate free histones remained the same in all the NaCl or MgCl 2 concentrations tested, indicating that these salts do not affect Gcn5p activity per se (Fig. 1A). In contrast, both nucleosome core particles and nucleosomal arrays were acetylated by rGcn5p only in the presence of a relatively narrow range of NaCl or MgCl 2 , with maximum acetylation observed in 50 mM NaCl or 2 mM MgCl 2 (Fig. 1, B and C, respectively). Under conditions utilized pre-viously by other laboratories, i.e. 0 or 100 mM monovalent cations, we also detected essentially no HAT activity using nucleosomal substrates (Fig. 1B). In addition, rGcn5p acetylated nucleosomal arrays in 50 mM KCl but not 50 mM KCl/50 mM NaCl (data not shown), indicating that the previously reported inability of rGcn5p to acetylate nucleosomal substrates was because of the use of non-optimal salt concentrations rather than an inhibitory effect of K ϩ . Finally, it is important to note that despite the lack of rGcn5p activity in Ն100 mM NaCl alone (Fig. 1B), acetylation of nucleosomes and nucleosomal arrays was observed in the presence of the physiologically relevant mixture of 150 mM NaCl and 2 mM MgCl 2 (data not shown).
Acetylation of nucleosome core particles and nucleosomal arrays by rGcn5p in 50 mM NaCl was very similar, although nucleosomal arrays appeared to be slightly preferred under these conditions (Fig. 1B). In contrast, rGcn5p acetylated nucleosomal arrays ϳ40% faster than nucleosome core particles in 2 mM MgCl 2 (Fig. 1C). The enhanced rates of acetylation of nucleosomal arrays relative to nucleosome core particles was strongly correlated with the degree of array folding; nucleosomal arrays were only partially folded in 50 mM NaCl and extensively folded in 2 mM MgCl 2 ( Fig. 1D; see Refs. 18 and 20), whereas nucleosome core particles sedimented at the expected value of 11 S (26) in both salt conditions (Fig. 1D, inset). Importantly, no salt-dependent histone dissociation occurred under the conditions used for the HAT assays as indicated by both the sedimentation data (Fig. 1D) and native agarose gel electrophoresis (data not shown). In addition, neither nucleosomes nor nucleosomal arrays were acetylated by rHat1p under comparable ionic conditions, indicating that the ability to acetylate nucleosomal substrates was rGcn5p-specific (data not shown). Taken together, the data in Fig. 1 demonstrate both that rGcn5p alone is capable of significantly acetylating nucleosomal substrates under optimal salt and substrate conditions and that rGcn5p more efficiently acetylates extensively folded nucleosomal arrays compared with partially folded arrays and nucleosome core particles.
Gcn5p Polyacetylates Free Histone H3 but Only Monoacetylates Nucleosomal H3-Acetylated free histones, nucleosome core particles, and nucleosomal arrays next were electrophoresed on an 18% SDS-polyacrylamide gel to determine which specific core histones had been modified by rGcn5p (Fig. 2). When free histones were incubated with rGcn5p, both H3 and H4 were acetylated in 50 mM NaCl (lane 1) and 2 mM MgCl 2 (lane 2), consistent with previous results (7,10,12,13). In contrast, under these conditions rGcn5p acetylated essentially only H3 in both nucleosomal arrays (lanes 5 and 6) and nucleosome core particles (lanes 9 and 10). In agreement with the liquid HAT assays (Fig. 1), the extent of H3 acetylation of nucleosomal arrays in 2 mM MgCl 2 was significantly greater than that observed for nucleosome core particles ( Fig. 2; lanes  6 and 10, respectively). No acetate incorporation was observed in the absence of rGcn5p. These results demonstrate that rGcn5p primarily modifies H3 in nucleosomal core particles and nucleosomal arrays.
Given that the assembly of histones into nucleosomes decreases the overall accessibility of the H3 N termini to rGcn5p (see Fig. 1, A-C), we investigated whether the acetylation target sites in the H3 N termini also were affected. Free histones, nucleosome core particles, and nucleosomal arrays were incubated with an excess of rGcn5p and acetyl-CoA in either 2 mM MgCl 2 or 50 mM NaCl for 0 -90 min and analyzed by AUT gel electrophoresis (24). The results obtained in 2 mM MgCl 2 are shown in Fig. 3. Under these conditions, the N termini of free histone H3 were polyacetylated. In contrast, only monoacety-lated H3 was observed in both core particles and nucleosomal arrays. No unacetylated H3 molecules remained after 90 min of incubation, indicating that all of the H3 N termini were either polyacetylated or monoacetylated in free histones or nucleosomal substrates, respectively (data not shown). Hence, when assembled into nucleosomes, only the total number of acetylatable sites within the H3 N termini were limited to acetylation by rGcn5p. Results obtained in 50 mM NaCl were similar to those in MgCl 2 (data not shown).
To determine the specific H3 residues acetylated in the three different substrates, microsequence analyses of the first 30 amino acid residues were performed (Fig. 3, C-E). The amount of radioactivity present in each cycle was determined by scintillation counting. Results indicated that polyacetylation of free H3 occurred at lysines 9, 14, and 18 (Fig. 3C). In contrast, H3 was acetylated only at lysine 14 in both nucleosomal core particles and nucleosomal arrays (Fig. 3, D and E). These data indicate that the structure and accessibility of the H3 N termini change upon assembly of free histones into nucleosomes and nucleosomal arrays. Recently, it has been reported that Gcn5p is required either directly or indirectly for the acetylation of residues 9, 14, and 18 of histone H3 in vivo (13,27). Our results are both consistent with these observations and extend them by showing that rGcn5p can directly acetylate all three residues of free H3 in the absence of other proteins in vitro.

DISCUSSION
The recruitment of the histone acetyltransferase, Gcn5p, to specific genes (16,27) and the ability of acetylation to decondense repressive higher order chromatin structures (28,29) have provided important clues into the molecular basis of how acetylation facilitates gene expression. Our results indicate that Gcn5p action and chromatin decondensation may be directly linked. Although it has been proposed that Gcn5p must interact with other protein subunits to acetylate nucleosomal arrays (10,(12)(13)(14)30), our studies have demonstrated unequivocally that rGcn5p per se is capable of acetylating both nucleosomes and nucleosomal arrays under appropriate ionic conditions, including those that approximate the ionic environment present in the nucleus. Furthermore, rGcn5p exhibits a marked preference for extensively folded nucleosomal arrays as FIG. 1. Histone acetyltransferase activity assays in NaCl or MgCl 2 . A, free core histones were incubated with rGcn5p in either in creasing NaCl (‚) or MgCl 2 (ƒ) concentrations as indicated in the figure for 20 min at room temperature. The amount of [ 3 H]acetate incorporation was ascertained by scintillation counting (see "Experimental Procedures"). Shown is the mean Ϯ S.E. of six independent experiments. B, nucleosome core particles () or nucleosomal arrays (OE) were incubated with rGcn5p in 0 -300 mM NaCl as in A. Plots were normalized to free histones to compensate for differences in enzyme activity between experiments. C, nucleosome core particles () or nucleosomal arrays (OE) were incubated with rGcn5p in 0 -10 mM MgCl 2 as in A. Plots were normalized to free histones to compensate for differences in enzyme activity between experiments. D, sedimentation velocity analysis of nucleosomal core particles and nucleosomal arrays in DB buffer with no salt (E), 50 mM NaCl (ƒ), or 2 mM MgCl 2 (‚). Samples were equilibrated at 20°C for 1 h and centrifuged at either 18,000 rpm for nucleosomal arrays or 45,000 rpm for nucleosomal core particles. Sedimentation coefficient distribution plots for nucleosomal core particles are shown in the inset.
FIG. 1-continued a substrate. Cumulatively, these observations necessitate several fundamental changes in the present model of how Gcn5p functions to regulate gene expression. Gcn5p is currently viewed as an inactive enzyme that is recruited to specific promoters through interaction(s) with transcriptional coactivators and when complexed subsequently leads to local acetylationdependent disruption of the structure of specifically targeted nucleosomes (16,27). The available data now indicate that Gcn5p both is capable of acetylating chromatin in the absence of coactivators and likely functions at the level of global chromosomal domains (i.e. Ն2-3 nucleosomes) by facilitating targeted chromatin decondensation. In support of the latter view, it is well established that extensively folded nucleosomal arrays are repressive to transcription by eukaryotic RNA polymerases (28,31,32), and it has recently been shown that an unexpectedly low threshold level of acetylation dramatically enhances transcription initiation and elongation by causing array decondensation (28). Importantly, studies performed with antibodies that specifically recognize histone H3 diacetylated at Lys-91 and Lys-14 (27) do not contradict a more global function for Gcn5p, because these experiments would not have detected any nucleosomes acetylated only at Lys-14 of H3 (Fig.  3) that were located away from the promoter.
As chromatin folds into progressively higher order structures with increasing salt concentrations, the N termini undergo significant changes in their location and accessibility (33,34). Hence, the most likely explanation for the ability of Gcn5ptranscription factor complexes to acetylate nucleosomal substrates in the absence of salt in vitro (7,10,13,25,30) is that the H3 N termini simply become more accessible under these conditions because of the presence of the Gcn5p-associated proteins. Although there is clearly a genetic relationship between Gcn5p and Ada proteins in yeast in vivo (35,36), the mechanistic roles of these accessory proteins remain to be fully defined. One function may be to potentiate the activity of Gcn5p (30). However, actions unrelated to inducement of Gcn5p catalytic activity, such as targeting to specific chromo-  somal loci or regulation of multiple acetylation site usage, also must be considered. In this regard, it should be noted that the mammalian Gcn5p has an extra ϳ350-amino acid N-terminal extension not found in the yeast homologue (37,38), which may serve to replace some or all of the functions contributed by the yeast accessory proteins.