reactive aldehydes , 
 such as acrolein and malondialdehyde ( mda ) , 
 react with dna and form exocyclic adducts .
acrolein , an ,-unsaturated 
 aldehyde commonly found in tobacco smoke and other exogenous sources ( petroleum industry waste , automobile exhaust , and overcooked 
 food ) is a mutagenic agent that has been implicated in the etiology of lung cancer .
acrolein is also formed endogenously as a byproduct of lipid peroxidation , alongside structurally related molecules such as mda . as a reactive 
 aldehyde
, acrolein condenses with deoxyguanosine ( dg ) through a michael 
 addition and subsequent cyclization to form two exocyclic 
 adducts : -oh - pdg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-]purin-10(3h)-one ) and -oh - pdg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-]purin-10(3h)-one ) ( figure 1 ) .
reactions of the environmental 
 pollutant acrolein and of the lipid 
 peroxidation byproduct malondialdehyde with dna guanine bases generate 
 the structurally similar exocyclic deoxyguanosine lesions -oh - pdg , 
 -oh - pdg , and m1dg .
dna adducts have deleterious biological 
 consequences 
 and are believed to underlie the mutagenic effects of acrolein and its ability to promote carcinogenesis .
-oh - pdg can block dna replication in human cells and can 
 cause g to a and g to t mutations .
by contrast , 
 -oh - pdg is efficiently bypassed by certain polymerases in both 
 bacterial and mammalian cells ; however , -oh - pdg can also form inter- and 
 intrastrand cross - links ( via an open - ring aldehydic form ) , which are difficult 
 to bypass and can cause mutations .
mda is an important 
 biomarker of lipid peroxidation , a deleterious 
 reaction between cellular lipids and reactive oxygen species produced 
 by inflammatory processes .
similar to acrolein , mda can also form an exocyclic dg adduct , m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)pyrimido[1,2-]purin-10(3h)-one ) .
this adduct is both a strong 
 block to replication and mutagenic , properties that may 
 contribute to inflammation - associated human malignancies , including 
 aging and cancer .
given the toxicity and mutagenicity of the acrolein- and mda - derived 
 exocyclic deoxyguanosine adducts , it is important to investigate if 
 these adducts are substrates for dna repair pathways . while the nucleotide 
 excision repair ( ner ) pathway can remove these lesions from dna , acrolein , the agent responsible for generating 
 the exocyclic dg lesions , inhibits ner .
little is known about the contribution of a direct reversal repair 
 pathway for removal of acrolein- and mda - derived exocyclic guanine 
 dna adducts .
alkb , the escherichia coli direct 
 reversal dna repair enzyme , can efficiently repair a wide range of 
 dna and rna alkyl lesions . as an fe(ii)- and -ketoglutarate - dependent dioxygenase , alkb 

uses molecular oxygen to oxidize and remove simple alkyl dna lesions 
 ( such as 3-methylcytosine , 1-methyladenine , 3-methylthymine , 6-methyladenine , 1- methylguanine ( m1 g ) , 2-methylguanine(m2 g ) , and 2-ethylguanine(e2 g ) ) and exocyclic 
 bridged lesions ( n1,n - ethenoadenine ( a ) , n1,n - ethanoadenine ( ea ) , 3,n - ethenocytosine ( c ) , hydroxy-3,n - ethanocytosine , and hydroxy-3,n - propanocytosine ) .
given the ability of alkb to remove alkyl groups from both n1 and n positions of guanine , we suspected that the exocyclic 
 guanine adducts -oh - pdg , -oh - pdg , and m1dg 
 could also be potential substrates for alkb .
this study investigated 
 the ability of alkb to repair the acrolein- 
 and mda - derived exocyclic dg lesions in single - straned ( ssdna ) or 
 double - stranded dna ( dsdna ) .
we established that all three exocyclic 
 dg lesions can exist in open - ring forms in the sequence context studied , 
 and we also investigated whether these open - ring forms are substrates 
 for alkb repair . using high - resolution quadrupole time - of - flight ( q - tof ) 
 ms , we found that alkb can oxidize all three exocyclic dg adducts , 
 in both the open- and closed - ring forms .
the alkb activity on the 
 lesions was more efficient in ssdna than in dsdna , with the base opposite 
 the lesion dictating the repair efficiency . among the three lesions 
 studied , -oh - pdg was most efficiently repaired , followed by 
 m1dg and -oh - pdg , respectively . by identifying and 
 characterizing the alkb reaction intermediates and products using 
 ms , ms / ms , and biochemical trapping experiments , a model for the chemical 
 mechanism of repair of exocyclic dg lesions by alkb is proposed .
the 
 observation that these important lesions are oxidized by alkb provides 
 evidence that direct reversal enzymes , such as alkb , may play important 
 roles as modulators of the biological consequences of acrolein and 
 mda .
oligonucleotide 16-mers of 
 sequence 5-gaagacctxggcgtcc-3 ( x = adduct ) 
 bearing exocyclic guanine adducts , -oh - pdg , -oh - pdg , and m1dg ( figure 1 ) were prepared 
 using the solid - phase methods and were 
 deprotected , purified , and characterized as described previously . 

table 1 shows the calculated mws of all the 
 oligonucleotides used in this study and their observed intermediates . 
 to determine the oligonucleotides concentrations , the extinction 
 coefficients (  ) of normal dna bases at 260 nm
alkb incubations were performed with the alkbn11 protein , 
 a version of alkb lacking the first 11 amino acids .
this truncated 
 protein has been previously shown to have activity similar to that 
 of wild - type alkb .
the alkbn11 
 protein was expressed in bl21(de3 ) cells and purified as described 
 previously .
the alkb incubations were 
 performed at 37 c for 2 h in a reaction buffer containing 45 
 mm hepes ( ph 8.0 ) , 0.9 mm -ketoglutarate , 67 m fe(nh4)2(so4)26h2o , and 1.8 mm ascorbate , followed by hplc / q - tof ms analysis . typically , 
 100 pmol dna was reacted with 5 m alkb ( or enzyme diluent for 
 control reactions ) in the presence of all cofactors in a 20 l 
 reaction volume .
the alkb incubations with double - stranded dna were 
 performed under similar conditions except that prior to alkb addition 
 1.2 equiv of complementary oligonucleotides were annealed by heating 
 the mixture at 65 c for 5 min .
the mixtures were cooled at a 
 rate of 0.1 c / s to 4 c and then used in alkb reactions 
 as described above .
the alkb repair 
 efficiencies were estimated from the fraction of starting material 
 converted into intermediates and products ( table 2 ) .
these values were determined by integrating the extracted 
 ion chromatogram ( eic ) peaks corresponding to each of the species 
 observed in the alkb reactions ( starting material , intermediates , 
 and products ) and calculating the percentage of each species formed 
 after alkb incubation relative to the total amount of material ( table 3 ) . in each reaction , the total amount of material 
 was calculated by summing the areas corresponding to the starting 
 material and all intermediates and products derived from the starting 
 material .
all of the values were background - corrected for the amount 
 of species ( intermediate or product ) that may have been present in 
 the alkb untreated control reactions . trapping 
 reactions of oligonucleotides with pfbha were performed in water at 
 room temperature for 1 h using 5 m dna and 500 m pfbha 
 in a 10 l incubation volume , followed by hplc / q - tof ms analysis , 
 as described previously . the calculated 
 mws of the trapped intermediates
melting 
 temperatures were 
 measured on the dsdna obtained by mixing the 16-mer oligonucleotides 
 containing lesions with equimolar amounts of the complementary oligonucleotides 
 containing each of the four possible natural bases ( c , t , g , and a ) 
 opposite the lesion site . the oligonucleotides ( 4 m in a total 
 volume of 25 l ) were mixed in alkb reaction buffer ( 45 mm hepes , 
 ph 8 , 0.9 mm -ketoglutarate , 1.8 mm ascorbate , 67 m 
 fe(ii ) ) supplemented with the high - resolution melting dye lcgreen 
 plus ( idaho technologies inc .
, salt lake city , ut ) to a final concentration 
 of 1. annealing was done by heating at 75 c for 5 min , 
 followed by a slow cooling ( 0.1 c / s ) to 4 c .
melting temperature , tm , was measured using a roche lightcycler 480 
 spectrophotometer , by heating the samples from 37 to 95 c at 
 a rate of 0.1 c / s and recording fluorescence five times every 
 degree celsius .
data analysis was performed using lightcycler480 software . 
 for each oligonucleotide and complement pair ,
reduction of exocylic dg lesions ( -oh - pdg , 
 -oh - pdg , and m1dg ) was performed by incubating 1 
 nmol of 16-mer oligonucleotide in 100 mm potassium phosphate , ph 8.0 , 
 with 1 m nabh4 for 1 h at 37 c in 10 l 
 reaction volume . to prevent excess pressure from hydrogen gas accumulation 
 from the nabh4 hydrolysis ,
subsequently , 
 the excess nabh4 and salts were removed by passing the 
 reaction mixtures through homemade dry - spun sephadex g50 fine ( amersham 
 biosciences ) columns .
after concentration by centrifugation under 
 vacuum , the purified reduced oligonucleotides were used in alkb reactions 
 using the conditions described previously .
all samples ( untreated , 
 nabh4 treated , and nabh4 then alkb treated ) 
 were analyzed using hplc - esi - tof mass spectrometry .
oligonucleotide analyses 
 were performed using agilent q - tof 6510 mass spectrometer ( palo alto , 
 ca ) setup as follows : needle voltage , 3.5 kv ; nitrogen drying gas , 
 8 l / min ; heated capillary , 340 c ; and nebulizer , 30 psig .
the 
 reaction mixtures were separated by hplc on a zorbax sb - aq column 
 ( 2.1  150 mm ; 3.5 m ; agilent technologies , palo alto , 
 ca ) at a 0.2 ml / min flow rate at room temperature .
solvents were 10 
 mm ammonium acetate in water ( a ) and 100% acetonitrile ( b ) .
lc  ms / ms analyses were performed on the agilent 6510 q - tof 
 instrument , operated in the negative ion mode with the following parameters : 
 gas temperature , 340 c ; esi capillary voltage , 3.5 kv ; nebulizer 
 pressure , 30 psi ; drying nitrogen gas , 8 l / min ; and fragmentation 
 energy , 3035 v. the theoretical fragmentation pattern of a 
 16-mer oligonucleotide is shown in figure 2a ; the expected monoisotopic masses of the fragments were calculated 
 using mongo oligo mass calculator , version 2.06 ( http://mods.rna.albany.edu/masspec-toolbox ) . a typical fragmentation spectrum ( e.g. , intermediate 3a/3b ) is shown in figure 2b .
the 
 comparison between the calculated and observed m / z values for the ms / ms spectrum of 3a/3b is shown in figure 2c .
ms / ms analyses 
 of the observable reactants , intermediates , and products for all of 
 the alkb reactions with 16-mer oligonucleotides containing -oh - pdg , 
 -oh - pdg , and m1dg are included in the supporting information ( figures s10s17 
 and tables s2s9 ) .
ms / ms analysis of prototypic 16-mer oligonucleotide 
 containing 
 the alkb - oxidized form of -oh - pdg .
( top ) predicted collision - induced 
 dissociation ( cid ) fragmentation pattern of the 16-mer oligonucleotide . 

( bottom ) 
 ms / ms fragmentation spectrum of 16-mer containing the alkb - oxidized 
 form of -oh - pdg .
a comparison of predicted and observed m / z values for the ms / ms fragmentation 
 pattern shown at the bottom of the figure is included in the supporting information ( table s6 ) .
the ability of alkb to repair 
 acrolein- and mda - derived exocyclic 
 dg adducts was measured by incubating site - specifically modified 16-mer 
 oligonucleotides with purified alkb protein .
following a 2 h incubation 
 at 37 c , the reaction mixtures were analyzed using high - resolution 
 ms .
for each lesion , experiments were conducted 
 in both the presence and absence of the alkb protein , with all of 
 the necessary cofactors .
figure 3 shows representative 
 ms spectra corresponding to each oligonucleotide containing an exocyclic 
 dg lesion before and after alkb treatment .
the molecular weight ( mw ) 
 of each of the 16-mer oligonucleotides employed was calculated , from 
 which the 4 charge monoisotopic mass ( all c , n , etc . ) was determined ( table 1 ) . 

for example , the mw of the 16-mer containing the -oh - pdg lesion 
 is 4960.88 ( da ) ; therefore , its monoisotopic 4 charge state 
 has a theoretical m / z of 1239.21 . 
 in this case ,
an m / z of 1239.20 
 was observed experimentally ( figure 3a ) , which 
 correlated well with the theoretical value ( table 1 ) . because the ms conditions used throughout this study produced 
 robust 4 charge states for all the oligonucleotides analyzed , 
 all of the m / z numbers discussed 
 below refer to 4 charge states , unless otherwise specified . 

the chemical structures corresponding to the peaks labeled in figure 3 as well as their proposed alkb - catalyzed transformations 
 are shown in figure 4 .
q - tof mass spectrometry 
 analysis of reactants and products of the 
 oligonucleotides containing exocyclic guanine lesions incubated with 
 alkb for 2 h. data represent the 4 charge envelopes ; multiple 
 ion mass peaks associated with each envelope reflect mostly the number 
 of c atoms in each 4 charge packet .
( a ) -oh - pdg ; ( b ) -oh - pdg + alkb ; 
 ( c ) -oh - pdg ; ( d ) -oh - pdg + alkb ; ( e ) m1dg ; 
 and ( f ) m1dg + alkb .
chemical structures and proposed pathways for alkb - mediated exocyclic 
 guanine lesions transformation : ( a ) -oh - pdg ( dotted border ) 
 and -oh - pdg ( solid border ) ; ( b ) m1dg ( dashed border ) .
interestingly , the mass spectrum 
 of -oh - pdg in the absence 
 of alkb showed two peaks : a major peak at m / z 1239.20 , which corresponds to the 16-mer containing the 
 -oh - pdg lesion ( structure 1a , calculated m / z 1239.21 ) , and a minor peak at m / z 1234.70 , which corresponds to a dehydrated 
 form of -oh - pdg ( structure 2 , calculated m / z 1234.71 ) ( figures 3a and 4a ) .
the chemical structure of 2 was confirmed by ms / ms fragmentation analysis ( figures s10s11 and tables s2s3 ) .
upon addition of alkb protein , two new products were observed 
 with m / z 1238.68 and 1243.18 ( figures 3b , s1a , and s2 ) .
the 
 product with mass 1243.18 was assigned to the oxidized form of the 
 parent -oh - pdg lesion ( structure 3a , calculated m / z 1243.21 ) , consistent with the 4 m / z units increase , which in the 4 
 charge state corresponds to 16 da , the mass of an oxygen atom .
the 
 product with mass 1238.68 is similarly 4 m / z units higher than the dehydrated form at 1234.70 , suggesting 
 that it could be an oxidized form of 2 ( structure 4 , calculated m / z 1238.71 ) 
 ( figures s1 and s2 ) .
however , structure 4 could also arise from dehydration of the oxidized intermediate 
 ( structure 3a ) , being 4.5 m / z units lower than the 1243.18 species ( which , in the 4 
 charge state , corresponds to the loss of a water molecule , 18 da ) . 

these data suggest that , under these reaction conditions , alkb can 
 oxidize the -oh - pdg lesion ( and its dehydrated form ) , transforming 
 it into the diol 3a that could spontaneously lose the 
 exocyclic bridge to restore the parental deoxyguanosine ( figure 4a ) .
however , the fully repaired deoxyguanosine product 
 was not observed , perhaps because the amount of oxidized intermediate 3a formed was relatively small , with only 5.5% conversion 
 ( table 3a ) .
the oligonucleotide containing 
 -oh - pdg had an experimental m / z of 1239.23 ( structure 5a , calculated m / z 1239.21 ) .
incubation 
 with alkb resulted in the conversion of about 60% of the -oh - pdg 
 starting material into four new species ( figure 3d and table 2 ) with m / z values of 1225.17 ( dg , structure 6 ) , 1243.17 
 ( diol intermediate 3a ) , 1238.67 ( single - dehydration product 4 ) , and 1234.17
relative mass differences between these peaks 
 ( figure s1b ) were used to propose chemical 
 structures for all of the intermediates ( figure 4a ) , which were further confirmed by an ms / ms fragmentation analysis 
 ( figures s12s14 and tables s4s6 ) .
the appearance of these intermediates and products suggests the 
 following reaction course for direct reversal of -oh - pdg damage : 
 alkb oxidizes the methylene group adjacent to the n - position of -oh - pdg to generate ,-dihydroxy - pdg 
 ( 3a ) , which can release mda to form undamaged guanine 
 ( 6 ) .
the final step of mda release from 3a is analogous to the release of glyoxal from the a glycol 
 intermediate that forms when alkb repairs the a lesion .
however , intermediate 3a can also 
 dehydrate to form 4 and , upon loss of another water molecule , 
 intermediate 7 , which is the mda adduct m1dg ( figure 4a ) . as will be shown later ,
however , given 
 the small amounts m1dg formed in the reaction of -oh - pdg 
 with alkb , the intermediates of the m1dg reaction with 
 alkb likely were below the limit of detection and therefore were not 
 observed .
when the oligonucleotide containing m1dg 
 ( starting material m / z of 1234.19 ) 
 was incubated with alkb 
 under similar conditions , four new mass envelopes were observed in 
 the mass spectrum ( figures 3f and s1c ) , besides the unreacted starting material . 

these peaks suggested a reaction course for direct reversal of m1dg damage by alkb ( figure 4b ) .
specifically , 
 the mass envelope at m / z 1238.19 
 was consistent with the alkb - catalyzed formation of an epoxide for 
 m1dg ( structure 8 in figure 4b , calculated m / z 1238.20 , 
 table 1 ) .
hydrolysis of the epoxide would generate 
 glycol 9 , observed at m / z 1242.69 , consistent with the calculated m / z 1242.71 .
the additional species observed at m / z 1247.19 indicated the presence of the trihydroxylated 
 intermediate 10 ( calculated m / z 1247.21 ) , which would be the hydration product of glycol 9 . the spontaneous loss of 2-hydroxy - mda from the trihydroxylated 
 intermediate 10 would generate the undamaged guanine 
 base , observed at m / z 1225.19 ( calculated m / z 1225.21 ) ( figures 3f and 4b ) .
the assignment of intermediates 
 was confirmed by ms / ms fragmentation analysis ( figures s15s17 and tables s7s9 ) .
alkb can repair 
 alkylated nucleic acid bases in both ssdna and 
 dsdna . while repair in the single - stranded context is typically more 
 efficient ,
there are examples of lesions 
 that are repaired by alkb equally well in ssdna and dsdna .
therefore , it was of interest to investigate 
 whether the acrolein- and mda - derived exocyclic dg lesions were also 
 substrates for alkb in dsdna .
alkb repair reactions were carried 
 out with each of the three exocyclic dg adducts opposite each of the 
 four canonical bases ( t , c , g , and a ) ( figures 
 s3s5 ) .
the amount of the starting material converted 
 by alkb ( table 2 ) was estimated by integrating 
 the extracted ion chromatograms ( eic ) of each peak in the mass spectra 
 ( see experimental procedures for details ) . 

likewise , the relative amounts of the intermediates and repair products 
 formed in each case were also estimated ( table 3a  c ) .
the data show that the efficiency of alkb - mediated oxidation 
 of exocyclic dg adducts depended not only on the type of lesion ( as 
 seen in ssdna ) but also on the nature of the base placed opposite 
 the lesion .
in general , the oxidation was less efficient in dsdna 
 compared with that in ssdna , with m1dg being a notable 
 exception , for which the maximum conversion of starting material was 
 observed in dsdna when placed opposite t ( table 2 ) . as a general trend ,
lesions were more efficiently converted when 
 placed opposite t than opposite any other base . the smallest conversion 

the quantification of intermediates and fully repaired 
 product 
 for each of the alkb reactions above provided some additional insights . 
 for -oh - pdg , the alkb conversion was at least 3-fold lower 
 in dsdna compared with that in ssdna , and the amount of diol intermediate 
 ( structure 3a ) formed in the dsdna reaction was reduced 
 by a similar amount .
however , the amount of fully repaired product , 
 dg , was disproportionally reduced ( by at least 10-fold with respect 
 to ssdna , table 3b ) , indicating that the final 
 step of mda release from 3a is impeded when alkb repair 
 occurs in dsdna . for m1dg opposite t ,
however , the amount of fully 
 repaired product was maximal in ssdna ( 8.1% ) compared with that in 
 dsdna ( 1.34.6% ) ( table 3c ) , suggesting , 
 as before , that the final step of the release of the oxidized exocyclic 
 bridge from intermediate 10 was impeded in dsdna . for 
 -oh - pdg in dsdna , alkb oxidation was even less efficient than 
 that in ssdna , with the oxidized intermediates detected at correspondingly 
 lower levels ( table 3a ) . just as in ssdna
, 
 no fully repaired dg product was observed in the alkb reaction of 
 -oh - pdg in dsdna .
however , it appears that the equilibrium 
 between the hydrated ( structure 1a ) and dehydrated ( structure 2 ) forms of -oh - pdg shifted toward the structure 1a in dsdna .
the reactions of alkb with dsdna consistently showed 
 that when the acrolein- and mda - derived exocyclic dg adducts were 
 placed opposite c the efficiency of repair was significantly diminished 
 ( table 2 ) .
we hypothesize that this effect 
 was due to the ability of exocyclic dg lesions to exist in open - ring 
 structures . in the case of -oh - pdg and m1dg ,
the presence 
 of the cognate base pairing partner ( dc ) in the opposite strand has 
 been shown to induce the opening of the exocyclic ring to expose the 
 watson 
crick base - pairing side of the damaged guanine and allow 
 it to form hydrogen bonds . to test this hypothesis in our sequence 
 context ,
the thermal melting temperatures ( tm ) of the oligonucleotides containing exocyclic dg adducts 
 opposite all four canonical bases were measured ( table 4 ) . consistent with this hypothesis and previous observations , the melting temperatures containing -oh - pdg and m1dg opposite c were the highest in each case ( as compared to other 
 complementary bases ) and approached the tm of a duplex containing a canonical g : c base pair at the lesion site . 
 when compared with a normal guanine , the presence of m1dg increased the melting temperature of the duplex when opposite 
 t , g , or a. this effect was likely due to the extended aromatic ring 
 system of m1dg , which allowed for additional favorable 
 stacking interactions .
by contrast with the other two lesions , the 
 oligonucleotide containing -oh - pdg was not stabilized opposite 
 any of the four canonical bases .
this observation was consistent with 
 the fact that the open - ring structure of -oh - pdg would allow 
 only the n position of guanine to interact 
 with the opposite base , while the n1 position would be still blocked .
the -oh - pdg and m1dg lesions have been reported to 
 equilibrate between ring - closed and ring - open forms . in dsdna ,
-oh - pdg 
 predominantly exists in open - ring form when placed opposite dc , whereas in ssdna , it exists mostly in the closed - ring 
 form . in both cases ,
-oh - pdg could be trapped by peptides , 
 with the conjugation being more effective in dsdna than in ssdna . the reversible , hydrolytic ring - opening of m1dg has also been well - studied , with the ring - open form of m1dg predominating in dsdna 
 when m1dg is placed opposite dc .
the presence of open - ring forms of the three exocyclic dg 
 lesions was studied in our sequence context by incubating the site - specifically 
 modified oligonucleotides used in the alkb repair studies with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine ( pfbha ) , a 
 nucleophilic alkoxyamine that forms stable oxime linkages with free 
 aldehydes .
after 1 h at room temperature , 
 the reaction mixtures were analyzed using high - resolution ms ( figure 5 ) .
the calculated and experimentally observed m / z values for the oligonucleotides trapped 
 with pfbha are listed in table s1 .
q - tof mass 
 spectrometry analysis of the pfbha trapping reactions 
 of oligonucleotides containing exocyclic guanine lesions .
( a ) -oh - pdg ; ( b ) -oh - pdg + pfbha ; ( c ) -oh - pdg ; 
 ( d ) -oh - pdg + pfbha ; ( e ) m1dg ; and ( f ) m1dg + pfbha .
all three oligonucleotides 
 containing the exocyclic dg lesions 
 were trapped by pfbha , indicating that they can potentially exist 
 in open - ring forms containing free aldehyde groups ( figure 5 ) . in the case of -oh - pdg ,
pfbha trapping 
 provides strong evidence that the exocyclic adduct ( 5a ) exists in equilibrium with an open - ring aldehyde form ( 5b ) . for m1dg ( 7 ) , however , the complete trapping 
 with pfbha does not unequivocally demonstrate the existence of the 
 ring - open aldehyde forms ( 11b or 12b ) , because 
 pfbha can react directly with the closed - ring form of m1dg and generate the oxime product without the formation of a free 
 aldehyde .
this alternative course of reaction is based on the observation 
 that the rate of ring opening of m1dg at neutral ph in 
 water is slower than the rate of trapping of m1dg with 
 hydroxylamine .
the presence of 
 pfbha completely trapped the oligonucleotide containing 
 -oh - pdg and m1dg .
the parent peak of -oh - pdg 
 at m / z 1239.21 ( calculated m / z 1239.21 ) shifted quantitatively to a pfbha - trapped species 
 at m / z 1287.97 ( calculated m / z 1287.97 ) .
similarly , the parent peak 
 of m1dg at m / z 1234.20 
 ( calculated m / z 1234.21 ) was completely 
 converted to a pfbha - trapped species at m / z 1287.47 ( calculated m / z 1287.46 ) . in the case of -oh - pdg , 1a ( observed m / z 1239.20 , calculated m / z 1239.21 ) can form an open - ring structure ( 1b ) that can be trapped with pfbha . however , as described 
 previously , 1a is also in equilibrium with the imine 2 , which could also react with pfbha to give a trapped product . 

only a partial trapping of the -oh - pdg was observed ( pfbha - trapped 
 species at m / z 1287.98 , calculated m / z 1287.97 ) , with the relative amounts 
 of untrapped oligonucleotides suggesting that pfbha reacted primarily 
 with 1b and not with 2 .
assuming pfbha trapping 
 of 1b is fast , these data also suggest that the formation 
 of the open - ring form of -oh - pdg is slower than that for -oh - pdg .
given the presence of the open - ring forms 
 of the three exocyclic dg lesions , it was of interest to investigate 
 whether alkb could also act on the open - ring forms .
sodium borohydride 
 ( nabh4 ) buffered with sodium phosphate was used to lock 
 the open - ring forms by reducing the open - ring aldehydes to alcohols , which were detected using high - resolution ms 
 ( figure 6 ) . for -oh - pdg , upon treatment 
 with nabh4 , the parent peak at m / z 1239.23
was quantitatively converted to a new peak at m / z 1239.71 , consistent with the addition 
 of two hydrogens . because there are no functional groups reducible 
 by nabh4 in the closed - ring structure of -oh - pdg , 
 or on any other normal base , the peak at m / z 1239.71 must correspond to the open - ring alcohol of -oh - pdg , 
 structure 16 ( calculated m / z 1239.72 ) ( figures 6b and s7 ) .
similarly , when the oligonucleotide containing -oh - pdg 
 was treated with nabh4 , the parent peak at m / z 1239.16 shifted to m / z 1239.67 ( figures 6a and s6 ) .
similar to the argument made for -oh - pdg , 
 the only functional group that could be reduced by nabh4 is the aldehyde of the open - ring form of -oh - pdg ( structure 1b , figure 4a ) to generate an open - ring 
 alcohol ( structure 19 , calculated m / z 1239.67 ) .
the dehydrated form of -oh - pdg ( structure 2 ) , a cyclic imine , was also reduced by nabh4 , 
 as suggested by the change in the isotopic distribution pattern and 
 the expansion of the peak envelope toward higher masses ( figure 6a )
. these changes are consistent with the appearance 
 of a peak at m / z 1235.16 ( figures 6a and s6 ) , which presumably 
 corresponded to a fully saturated 1,n - propano - dg ( structure 15 , calculated m / z 1235.21 ) ( figure 6a ) . 

both reduction products ( 19 and 15 ) are 
 consistent with the reported nabh4 reduction of the -oh - pdg 
 free nucleoside .
additional evidence 
 for the formation of 15 was obtained by running the nabh4 reaction at a lower ph ( 5.8 instead of 7.0 ) , which improved 
 the yield of reduction of 2 ( figure 
 s9 ) . however , even at low ph , reduction of 2 was 
 not quantitative , perhaps due to the lesser reactivity of the aromatically 
 stabilized cyclic imine toward nabh4 compared to that of 
 an aldehyde .
q - tof mass spectrometry analysis of the reactions of oligonucleotides 
 containing exocyclic guanine lesions with nabh4 and the 
 subsequent incubation of the reduced forms with alkb for 2 h. each 
 peak is labeled with its monoisotopic m / z value , in the 4 charge state . the corresponding chemical 
 structures and
( a ) ( top ) -oh - pdg , ( middle ) 
 -oh - pdg + nabh4 , ( bottom ) -oh - pdg + nabh4 + alkb .
( b ) ( top ) -oh - pdg , ( middle ) -oh - pdg 
 + nabh4 , ( bottom ) -oh - pdg + nabh4 + alkb . 

( c ) ( top ) m1dg , ( middle ) m1dg + nabh4 , ( bottom ) m1dg + nabh4 + alkb .
when exposed to nabh4 , the m1dg - containing 
 oligonucleotide ( observed m / z 1234.19 , 
 calculated m / z 1234.21 ) was converted 
 to a reduced species detected at m / z 1234.70 ( figures 6c and s8 ) .
the reduced ring - open form of m1dg requires the initial 
 addition of a water molecule to produce 11a or 12a ( calculated m / z 1238.71 ) 
 ( figure 4b ) , which could subsequently be reduced 
 with nabh4 .
however , the open - ring form of m1dg is known to enolize readily and thus is not very reactive toward 
 nabh4 .
consistent with this 
 fact , a product with an m / z of 1239.21 
 corresponding to a reduced version of 11a or 12a could not be detected ( figures 6c and s8 ) ; therefore , under the reaction conditions , 
 m1dg could not be trapped as an open - ring species by nabh4 .
nevertheless , the reaction of alkb with the reduced form 
 of m1dg provided additional insights into the regioselectivity 
 of the alkb repair reactions on exocyclic dg structures .
after 
 nabh4 treatment , the oligonucleotides were first 
 purified using sephadex g-50 columns to remove excess nabh4 and other salts and were then allowed to react with alkb under the 
 same conditions as those previously described .
alkb produced more 
 fully repaired product ( dg ) when incubated with nabh4-treated 
 lesions as compared to the incubations with untreated lesions . for 
 -oh - pdg , these reaction conditions produced detectable levels 
 of fully repaired dg ( 6 ) .
the amount of the reduced open - ring 
 alcohol 19 did not change upon treatment with alkb , whereas 
 the amount of 15 was diminished in the alkb reaction 
 ( figure 6a ) .
these observations indicated that 
 the fully repaired dg was not generated from the reduced open - ring 
 form 19 , but rather from the 1,n - propano - dg reduced intermediate ( 15 ) via two 
 successive oxidations by alkb , which would generate 3a ( figure 6a ) .
for the reduced ring - open 
 form of -oh - pdg ( 16 ) , the alkb reaction intermediates 
 were consistent with oxidation 
 at the carbon attached to the n position 
 of guanine with subsequent release of a three - carbon aldehyde to generate 
 fully repaired dg ( figure 6b ) .
this reaction 
 was very efficient , with most of the starting material being consumed 
 along with a high yield ( 50% ) of dg . for nabh4-treated 
 m1dg ,
the alkb reaction generated several intermediates 
 ( figure 6c ) , consistent with oxidation of 18 at the carbon attached to the n position of guanine to generate 12a , which upon dehydration 
 yielded the original m1dg lesion ( structure 7 , observed m / z 1234.23 ) .
subsequently , 
 m1dg ( 7 ) reacted with alkb , as shown before 
 ( figures 3f and 4b ) , 
 to generate epoxide 8 , triol 10 , and , upon 
 spontaneous loss of 2-hydroxy - mda , the fully repaired product , dg . 
 in an alternative reaction course
, 18 could react with 
 alkb to generate epoxide 20 and upon hydration , diol 21 ( figure 6c ) .
subsequently , 21 can be further oxidized by alkb to generate triol 10 , leading to additional repair product dg .
these additional 
 reaction pathways could explain the improved yield of fully repaired 
 product , dg , when nabh4-treated m1dg was incubated 
 with alkb .
the three exocyclic dg 
 lesions investigated ( i.e. , -oh - pdg , 
 -oh - pdg , and m1dg ) are important biomarkers of exposure 
 to ubiquitous environmental chemicals and 
 inflammation - induced lipid peroxidation byproducts , acrolein and mda . the present study demonstrates 
 that the exocyclic dg lesions are processed by alkb by a complex but 
 chemically understandable biochemical pathway . among the three lesions 
 studied in ssdna , -oh - pdg was repaired most efficiently , followed 
 by m1dg and -oh - pdg ( table 2 and table 3a  c ) .
the overall alkb 
 efficiency at repairing the lesions was generally lower in dsdna than 
 that in ssdna , as evidenced by the lower amounts of fully repaired 
 product formed in each case ( table 3a  c ) . 
 however , in the one case , when m1dg was opposite t , the 
 total amount of starting material consumed was higher than in the 
 incubation of alkb with m1dg in ssdna .
the base placed 
 opposite the lesion also influenced the repair reactions . generally , 
 when the lesions were placed opposite t , the repair was most efficient , 
 with c being the opposite base that gave the lowest conversions ( tables 2 and 3a  c ) .
these 
 observations can be rationalized by the base - flipping mechanism proposed 
 for alkb , in which a mispaired base ( i.e. , 
 lesion opposite t ) is more likely to splay out into the enzyme active 
 site to be repaired .
by contrast , when the exocyclic dg lesions are 
 placed opposite c , a  correct base pair  is formed ( in 
 the case of -oh - pdg and m1dg , via their open - ring 
 forms ) , which hinders base - flipping , effectively hiding the lesion 
 from alkb and reducing the repair efficiency .
this mechanism is consistent 
 with our previous study of alkb activity on n - dg alkyl lesions , such as m2 g , 
 e2 g , and the bulkier n - furfuryl - dg and n - tetrahydrofurfuryl - dg , which are good alkb 
 substrates in ssdna but are poorly repaired when placed in dsdna opposite 
 dc .
furthermore , the preference of alkb 
 for repairing exocyclic dg lesions in ssdna suggests that transcriptionally 
 active regions of the genome are likely to be most protected by alkb 
 activity , which complements other repair pathways that target exocyclic 
 dg lesions only in dsdna contexts ( i.e. , ner ) .
all three exocyclic dg lesions studied have been 
 proposed to exist 
 in equilibrium with open - ring forms .
the trapping of the -oh - pdg and -oh - pdg 
 with pfbha ( figure 5 ) suggests that there is 
 a low but kinetically useful concentration of the ring - opened aldehyde 
 form in equilibrium with the closed - ring form .
subsequently , by using 
 nabh4 to reduce the aldehydes and trap the open - ring forms 
 of -oh - pdg and -oh - pdg , the activity of alkb on the 
 open - ring forms was also investigated .
n-(3-hydroxypropyl)-g , the nabh4-reduced open - ring form 
 of -oh - pdg , was shown to be a good substrate for alkb repair , 
 even better than the closed - ring -oh - pdg substrate ( figure 6b ) .
the reduced open - ring form of -oh - pdg , 
 n1-(3-hydroxypropyl)-g , was not significantly oxidized by alkb ; instead , 
 the reduced version of the dehydrated -oh - pdg , 1,n - propano - dg ( structure 15 ) , reacted with 
 alkb ( figure 6a ) . for m1dg
, 
 however , the pfbha trapping does not necessarily 
 support the existence of the ring - opened aldehyde form , because the 
 trapped product can also be formed by a direct reaction of pfbha amine 
 at the carbon adjacent to n1 of m1dg .
consistent with this argument , m1dg incubation with nabh4 did not generate a measurable amount of trapped open - ring 
 forms ; the reduction yielded the closed - ring product 18 , which was also a good substrates for alkb , on par with the parent 
 species m1dg . taken together , the trapping experiments 
 demonstrated that alkb can process both open - ring species ( such as 
 the open - ring of -oh - pdg ) and closed - ring species ( such as 
 the ones derived from m1dg ) .
however , due to equilibria 
 between open- and closed - ring forms , the alkb repair reactions of 
 exocyclic dg lesions are complex , involving overlapping pathways with 
 multiple convergence points ( figure 4 ) . given the complexity of the repair pathways ( figure 4 ) , it is challenging to establish unambiguously which reaction 
 pathway ( i.e. , oxidation of open- or closed - ring intermediates ) is 
 predominant .
specifically , is alkb processing the exocyclic dg lesions 
 by directly oxidizing the closed - ring forms or is alkb reacting primarily 
 with the open - ring forms , essentially simpler n1- and n - alkyl guanines that are known to be alkb substrates ? 
 for -oh - pdg ,
the data were more consistent with the alkb oxidation 
 on the closed - ring form .
when this substrate was reduced to the open - ring 
 form with nabh4 , no corresponding alkb oxidation intermediate 
 was detected ; the alkb reaction presumably occurred only with the 
 reduced dehydrated version of -oh - pdg ( structure 2 ) , which is a closed - ring form ( figure 6a ) . 
 a possible explanation for this preference may be that the open - ring 
 form of -oh - pdg generated a bulky alkyl group on the n1 position 
 of guanine , which did not fit in the active site of alkb , unlike a 
 smaller n1-methyl - dg substituent , which is repaired by alkb .
for -oh - pdg , the data suggest that 
 both the closed- and 
 open - ring forms could be substrates for alkb .
we demonstrated that 
 alkb more efficiently repaired the reduced , open - ring form of -oh - pdg 
 than the unreduced , closed - ring -oh - pdg lesion .
this observation 
 is consistent with previous work , which demonstrated that alkb can 
 repair guanines substituted at n positions 
 with alkyl groups of varying sizes , including bulky alkyl substitutents .
it is noted , however , that the observed repair 
 of the open - ring form does not rule out the possibility that alkb 
 can additionally oxidize the closed - ring form of -oh - pdg ( i.e. , 
 the 5a to 3a step of figure 4a ) .
the data on the alkb repair of m1dg lesion 
 are most 
 consistent with the formation of an epoxide intermediate ( 8) of the closed - ring form , which subsequently gets hydrated twice 
 to form triol 10 .
intermediate 10 can also 
 be obtained from the oxidation and hydration of intermediates 11a or 12a , which are hydrated forms of m1dg .
however , the presence of these hydrated forms was not 
 detectable in the starting material , and their reduced forms were 
 not observed in the nabh4 reaction ( figure 6c ) .
therefore , the alternative pathways involving 11a and 12a are expected to play a minor role , if any , 
 in the alkb reaction ( figure 4b ) .
the data 
 on m1dg repair by alkb suggest that the enzyme , similar 
 to its mechanism on a , primarily acts on the closed - ring form 
 of the substrate .
the alkb repair data on exocyclic dg lesions 
 also provided some 
 insight on the alkb selectivity between oxidation at n1- or n - attached carbons , in the context of an exocyclic 
 propano adduct . by placing the three - carbon exocycle into
a published 
 alkb structure , it was determined that 
 the exocyclic carbon atoms adjacent to the n1 and n positions are situated at similar distances relative 
 to the iron center of alkb ( figure s19 ) . 

this suggests that alkb , in principle , should oxidize an exocyclic 
 propano adduct equally well at either the n1 or n position .
however , in the present study , alkb repaired 
 the two oh - pdg lesions with very different efficiencies ( table 2 ) , with -oh - pdg being repaired more efficiently 
 than -oh - pdg .
one explanation for the different alkb reactivity 
 toward these isomeric lesions may be the propensity to form open - ring 
 species .
the open - ring form of -oh - pdg shifts the entire alkyl 
 substituent to the n1 position , which likely can not be accommodated 
 in the alkb active site ; by contrast , the open - ring form of -oh - pdg 
 contains the alkyl substituent at n , 
 which can swivel away from the protein to potentially reduce steric 
 clashes . on the basis of these considerations ,
we speculate that the 
 most likely reaction course of the fully reduced exocyclic propano - dg 
 ( 16 ) with alkb involves oxidations first at the n1 carbon 
 ( -position ) and subsequently at the n carbon ( -position ) to generate the fully repaired 
 dg .
compared with other known exocyclic alkb substrates , such 
 as a 
 and ea , the acrolein- and mda - derived dg lesions are repaired less 
 efficiently under similar conditions .
one possible explanation is that three - carbon bridges 
 are bulkier than two - carbon bridges and some of them ( i.e. , -oh - pdg , 
 -oh - pdg ) can form puckered structures that are harder to accommodate 
 in the active site of the enzyme . furthermore , the position of a three - carbon 
 exocycle between n1 and n of guanine 
 could be suboptimal in the alkb active site compared with the two - carbon 
 bridge between n and n1 of adenine .
finally , alkb has 
 been demonstrated to prefer alkylated adenines compared to alkylated 
 guanines ; specifically , n1-methyl - adenine is repaired significantly 
 more efficiently than n1-methyl - guanine .
this difference in reactivity is believed to be due to the fact 
 that n1-methyl - adenine has a positive charge on n1 , whereas n1-methyl - guanine 
 is a neutral species .
proposed that , in general , 
 the preference of alkb toward positively charged lesions is likely 
 due to the presence of negatively charged aspartate residue ( asp-135 ) 
 in the active site .
therefore , it is 
 not surprising that this trend extends to exocyclic alkyl lesions , 
 where the exocyclic guanines in the present study are repaired less 
 efficiently by alkb than exocyclic adenines , in part because they 
 are neutral species .
furthermore , the higher repair efficiency of 
 alkb when acting on positively charged lesions could also be explained 
 by the propensity of the alkb oxidation intermediates of such lesions 
 to decompose and generate the fully repaired base .
consequently , it 
 is anticipated that alkb oxidation intermediates with pka s of the base nitrogens altered in a direction 
 that favors protonation would convert more efficiently to the fully 
 repaired product .
however , in the case of the exocyclic dg lesions , 
 the alkb oxidation intermediates are generally more abundant than 
 fully repaired dg , suggesting that the pka s of the n1 or n nitrogen of 
 the intermediates are not altered significantly during the alkb reactions .
the present work established the chemical competence of the e. coli dioxygenase alkb to oxidize and repair acrolein- 
 and mda - derived exocyclic guanine lesions , suggesting that one role 
 of alkb may be to alleviate the mutagenic and toxic consequences of 
 reactive aldehydes derived from these primary toxicants .
additionally , 
 the ability of alkb to repair the open - ring forms of exocyclic guanine 
 lesions suggests that alkb may also modulate the toxicity and mutagenicity 
 associated with the formation of inter- and intrastrand cross - links generated by the open - ring forms of the acrolein- and mda - derived 
 exocyclic guanine lesions .
given that alkb has nine mammalian homologues ( abh18 , fto ) , it is anticipated that these repair pathways 
 may also be operating in mammalian cells ; however , the extent to which 
 these pathways play a role in modulating the mutagenic and carcinogenic 
 risk associated with exposure to acrolein and inflammation - derived 
 mda is not known and remains to be established .
 the 
 structurally related exocyclic guanine adducts -hydroxypropano - dg 
 ( -oh - pdg ) , -hydroxypropano - dg ( -oh - pdg ) , and 
 m1dg are formed when dna is exposed to the reactive aldehydes 
 acrolein and malondialdehyde ( mda ) . 
 these lesions are believed to 
 form the basis for the observed cytotoxicity and mutagenicity of acrolein 
 and mda . in an effort to understand the enzymatic pathways and chemical 
 mechanisms that are involved in the repair of acrolein- and mda - induced 
 dna damage , we investigated the ability of the dna repair enzyme alkb , 
 an -ketoglutarate / fe(ii ) dependent dioxygenase , to process 
 -oh - pdg , -oh - pdg , and m1dg in both single- 
 and double - stranded dna contexts . by monitoring the repair reactions 
 using quadrupole time - of - flight ( q - tof ) mass spectrometry , 
 it was 
 established that alkb can oxidatively dealkylate -oh - pdg most 
 efficiently , followed by m1dg and -oh - pdg . 
 the alkb 
 repair mechanism involved multiple intermediates and complex , overlapping 
 repair pathways . 
 for example , the three exocyclic guanine adducts 
 were shown to be in equilibrium with open - ring aldehydic forms , which 
 were trapped using ( pentafluorobenzyl)hydroxylamine ( pfbha ) or nabh4 . 
 alkb repaired the trapped open - ring form of -oh - pdg 
 but not the trapped open - ring of -oh - pdg . 
 taken together , this 
 study provides a detailed mechanism by which three - carbon bridge exocyclic 
 guanine adducts can be processed by alkb and suggests an important 
 role for the alkb family of dioxygenases in protecting against the 
 deleterious biological consequences of acrolein and mda . 