Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.
Accidental DNA double strand breaks (DSBs) arise during unperturbed proliferation. Such “endogenous” or “spontaneous” chromosome breakage does not occur randomly throughout the genome, but at specific loci, often referred to as fragile sites. Fragile sites have been observed in organisms ranging from bacteria to mammals, suggesting that they might be a ubiquitous feature of the genome [1], [2], [3], [4]. Evidence points to the existence of multiple types of fragile sites that are distinguishable from one another based on its structure, function, and/or genetic requirement (s) for its stability [5], [6], [7]. Mammalian fragile sites are one of the most extensively studied naturally occurring breakage prone regions of the genome. They are classified as either “rare” or “common”, depending on their incidence among general population [4]. Rare fragile sites are found in less than 5% of the population. In most cases, rare fragile sites are tri-nucleotide repeats, expansion of which has been linked to conditions such as Fragile X-syndrome and Huntington disease [8]. In contrast, common fragile sites are present in all individuals and can be found on every chromosome, indicating that they are a normal component of the chromosome. Common fragile sites extend over large regions of the genome, from several hundred kilobases (kb) to over 1 megabase (Mb) with breaks or gaps occurring throughout these regions. There is no sequence determinant that defines common fragile sites [4], [9]. Studies have implicated a link between common fragile sites, genome instability, and cancer [9]. For instance, some fragile sites have been shown to be loci of frequent chromosome deletions, translocations, and/or viral genome integration [10], [11] as well as oncogenic chromosomal rearrangements (e. g. [12]). Combining these with the observations that some fragile sites in model organisms exhibit elevated rates of recombination, it has been proposed that mammalian common fragile sites are recombination hotspots and that increased recombination activities at these loci contribute to cancer. Replication Slow Zone (RSZ) is a type of fragile site in budding yeast and a putative homolog of the mammalian common fragile sites. It was identified as loci of preferred chromosome breakage following inactivation of Mec1, the budding yeast homolog of ATR, where high levels of replication dependent single stranded DNA (ssDNA), a precursor to DSBs, accumulate [3], [13]. Like its mammalian counterpart, RSZs are relatively large (∼10 kb), and appear to be a normal component of the chromosome. Other similarities between the two include; (i) timing of their replication during normal S-phase, which occurs late, (ii) their sensitivity to mild replication stress and inactivation of the ATR/ATM family kinases, and (iii) the lack of a defining sequence determinant (s) [3], [14], [15], [16]. The mammalian ATR/ATM and their budding yeast homologs Mec1/Tel1 are conserved signal transduction proteins best known for their roles in S-phase and DNA damage checkpoint responses [17]. In addition, they also play essential roles in a number of fundamental DNA and chromosomal processes including genome duplication, meiotic recombination, and DNA repair (e. g. [3], [18], [19]). Here, we utilized RSZ as a model to test the proposal that mammalian common fragile sites are recombination hotspots. Unexpectedly, we found that recombination rates at RSZ and non RSZ loci were comparable under all conditions tested, indicating that RSZs are not recombination hotspots. Based on these and other observations, we propose a model whereby regulation of the nature of the recombination outcome (s), irrespective of the overall recombination activity, may play a key role in controlling genome rearrangements.
The observations that stalled- and collapsed- replication forks can promote recombination [6], [7], [25], [26] and that RSZs are loci of slowed replication fork progression during normal S-phase [3], suggest that RSZs might be recombination hotspots during normal proliferation. (NB: In the current context, an arrested fork that can ultimately resume replication without intervention is referred to as a “stalled fork” whereas that requires an active fork-restart process is referred to as a “collapsed fork”). To test this, we compared the rate of URA3 inactivation at the RSZ and non RSZ loci under a standard yeast growth condition (2% glucose at 30°C, hereupon referred to as “YPD”). For each locus, the rate was estimated by the method of the median from two independently derived strains, and the average of the two was used for the locus-to-locus comparison (Figure S2) [24]. In haploids, the locus specific recombination rates varied very little, from 1. 8×10−5 per cell generation at ORI to 2. 1×10−5 at RSZ1 (Figure 2Ai). In diploids, the variation was greater, about 3 fold, and ranged from 1. 1×10−5 per cell generation at TER to 3. 3×10−5 at NON (Figure 2Aii). Importantly, the rate of URA3 inactivation at the two RSZs was not higher than the three non RSZ loci in either haploids or diploids. We conclude that RSZs are not spontaneous recombination hotspots, defined as loci of intrinsically higher recombination activities. Chromosome breakage at RSZs does not occur during normal proliferation, but is promoted by a modest level of HU, high temperature, and/or inactivation of Mec1 [3], [21]. Thus, it is possible that the putative recombination hotspot activity associated with RSZs might also require these conditions. To test this, we examined the effect of HU. The same ten haploid and ten diploid strains analyzed above were subjected to a transient (18 hour) exposure to 10 mM HU before 5FOA selection. We found that the exposure lead to a statistically significant increase in rate of URA3 inactivation at every locus (Figure 2D). Importantly, the rates at the two RSZs were not any higher than the three non RSZ loci in either haploids or diploids (Figure 2B). We conclude that RSZs are not recombination hotspots even under a condition that promotes RSZ specific chromosome breakage. Next, we examined the nature of genetic alternations associated with URA3 inactivation in diploids. To this end, we utilized Southern Blot analysis that enabled us to monitor the presence of the following three alleles (Figure 3A, Figure S3); (i) WT, (ii) the allele containing the hisG-URA3-hisG reporter integrated, hereupon referred to as “INT” for “integrant”, and (iii) the “pop-out” or “PO” allele, indicative of an intrachromatid or intersister (IC/IS) recombination or recombination related event (Figure 1C). As expected, the parent ura3 diploid strain exhibited a single band diagnostic of the WT allele at each of the five loci examined; in contrast, each of the URA3 heterozygotes derived from the parent exhibited an additional band (s), corresponding to the INT allele (Figure 3B, Figure S3). We found that all 5FOAR colonies examined had lost the INT fragment, suggesting that URA3 inactivation in every case involved a relatively large structural change (s) at the integration locus (Figure 3C; data not shown). Overall, ∼80% (117/146) of the samples exhibited the diagnostic PO band, indicating that on average, URA3 inactivation was four times more likely to occur via an IC/IS-mediated event than all other mechanisms combined (Figure 3E “Total”). The latter is consistent with previous reports on strong IC/IS-bias in mitotic recombination (e. g. [27]). Locus specific PO fraction ranged from 70% (14/20) at TER to 100% (26/26) at RSZ2, suggesting that the extent of IC/IS bias might be influenced by local environment (Figure 3E). The average PO fraction for the two RSZs was higher than the three non-RSZ loci (90% versus 73%; Figure 3E); however the difference was not statistically significant (p = 0. 1209). We conclude that recombination activity at RSZs during standard growth condition is comparable to non RSZ loci with regard to both the rate of recombination and the extent of the IC/IS bias. The same Southern Blot analysis was performed on 5FOAR colonies that arose in the presence of HU. Similarly to the YPD samples, all 148 HU 5FOAR samples had lost the diagnostic INT fragment (Figure 3D; data not shown). Overall, 53% (79/148) of the HU 5FOAR colonies carried the PO allele, a significant reduction from the 80% (117/148) observed in YPD (Figure 3F “Total”; Chi square test, p<0. 0001). The negative effect of HU on IC/IS bias was observed at every locus; however, the only statistically significant reduction was at the two RSZs (Fisher' s exact test; Figure 3F). At RSZ1, the PO fraction was reduced from 80%in YPD to 33% in HU, indicating that the 4∶1 bias toward IC/IS mediated URA3 inactivation in YPD was completely lost in HU, where the majority of URA3 inactivation occurred via non IC/IS mediated events. RSZ2 was notable because the extent of IC/IS bias in YPD appeared to be unusually strong (26/26; Figure 3E). In HU, we found that six of 29 had undergone a non IC/IS mechanism of URA3 loss, suggesting that the negative effect of HU might be irrespective of the intrinsic robustness of the IC/IS-bias. In the current analysis, a 5FOAR colony exhibiting only the WT band (e. g. Figure 3CD, samples denoted by an “*”) was inferred to have undergone URA3 inactivation via a non IC/IS mediated event, such as an IH gene conversion/crossover, ectopic recombination (ECT), or chromosome loss (Figure 1C). As mentioned above, it is possible to distinguish an IH event from the others by the virtue of the fact that a 5FOAR colony that arose via an IH recombination event would carry two copies of WT allele, while the rest carries just one (Figure 1C). Indeed, quantitative analysis of the WT fragments in Southern Blot images revealed that some of the non IC/IS samples had a two-fold greater signal associated with the WT band relative to an IC/IS sample that carried a copy of the WT and the PO allele each (Figure 3G, compare WT band intensity in “IH” and “IC/IS” lanes); these samples were inferred to have undergone URA3 inactivation via IH-recombination. We were also able to confirm the occurrence of an ectopic (ECT) event by the presence of a novel chromosome sized fragment containing the hisG sequence on a Southern Blot of a pulse field gel (PFG) (e. g. Figure 3F), suggestive of a chromosome translocation event. Applying these criteria, we were able to infer that the majority of 5FOAR colonies that arose in YPD (85/86) or in HU (78/89) had undergone URA3 inactivation via one of these three (IC/IS, IH, or ECT) mechanisms (Figure 3I and J). The rest, 1/86 in YPD and 11/89 in HU, was classified as “Other”, which would include mechanisms such as chromosome loss. Of the 86 YPD 5FOAR colonies analyzed, about 25%, or 15, had undergone URA3 loss via a non IC/IS event (s). Among them, all but two were mediated by IH recombination. The remaining two corresponded to an ECT and an Other event each (Figure 3I, “Total”). In HU, a reduction in the fraction of IC/IS event was accompanied by a significant increase in the fractions of ECT and Other, where they rose to 8% (7/89) and 12% (11/89) from about 1% in YPD (Figure 3J, “Total”). The only significant increase in IH fraction conferred by HU was at RSZ2, where it rose from 0/26 in YPD to 6/29 in HU (Figure 3I and J). To test whether the effect of HU on the IC/IS bias might have been due to reduced dNTP levels, we examined the effect of sml1Δ. Sml1 is a negative regulator of RNR and its deletion leads to a ∼2. 5 fold increase in dNTP levels [22]. We found that the mutation conferred a modest, but statistically significant increase in the overall PO fraction from 80% (117/146) in YPD to 89% (89/100) in sml1Δ (Chi-square analysis, p<0. 05; Figure 4F, “Total”). The observed effects of HU and sml1Δ suggest a positive correlation between dNTP levels and the extent of IC/IS bias, and implicate dNTP availability in regulation of recombination outcomes and genome rearrangements. The results also revealed that the effect of sml1Δ on IC/IS bias might be locus dependent; while the overall effect was an increase, the mutation actually lead to a modest decrease in the bias at RSZ2 from 100% (26/26) in WT to 90% (18/20) in sml1Δ (Figure 3E; Figure 4F). Similarly, we found that the effect of sml1Δ on recombination rate was locus specific; while the mutation lead to an increase at RSZ1, RSZ2, and TER, but it reduced the rate at ORI (Figure 4D, E). Overall, the effect of sml1Δ on ORI differed from the rest in that it was the only locus where the mutation conferred a statistically significant reduction in recombination rate and a significant increase in the IC/IS-bias. It would be require analysis of additional origin sequences to confirm whether the observed effect might be origin specific. No significant effect of sml1Δ was observed on the rate of URA3 inactivation in haploids (Figure 4A, C). If chromosome breakage is mechanistically linked to recombination activity, then HU, a condition that promotes RSZ specific chromosome breakage, should also confer an RSZ specific increase in the rate of URA3 inactivation. Unexpectedly however, we found that HU increased the rate at both RSZ and non RSZ loci, indicating that the breakage was not linked to recombination activity. To confirm this further, we examined the effects of additional conditions shown to regulate chromosome breakage at RSZs. Specifically, we chose high temperature and mec1Δ sml1Δ, the two conditions shown to promote the breakage, and rrm3Δ, a mutation shown to suppress it [21]. Rrm3 encodes a DNA helicase involved in replication fork progression through ∼1000 discrete fork pause sites in the budding yeast genome, whose inactivation leads to fork stalling at these loci [28], [29]. The rrm3Δ mediated fork stalling triggers Mec1/Tel1 dependent S phase checkpoint activation and Sml1 removal. The latter in turn, promotes fork progression through RSZ and prevents RSZ breakage even in the absence of Mec1 function [21], [30]. We limited our analyses to diploids only, where the effect on IC/IS bias can be assessed in addition to the rate of URA3 inactivation. Results showed that high temperature (37°C), rrm3Δ, and mec1Δ sml1Δ increased the average rate of URA3 inactivation by ∼2. 5 fold over WT at 30°C (Figure 5A, “Average”). Low temperature (23°C), on the other hand, lead to ∼30% reduction. These observations confirmed previous reports on recombinogenic effects of rrm3Δ and mec1Δ sml1Δ. Furthermore, they revealed a positive correlation between temperature and recombination rate, suggesting that temperature itself might regulate endogenous recombination activity. Importantly, the observed effect was not specific to RSZs, providing further support for the notion that chromosome fragility and recombination activity at RSZs are not mechanistically linked. Similarly to HU, the three recombinogenic conditions, 37°C, rrm3Δ, and mec1Δ sml1Δ lead to a reduction in the overall PO fraction (Figure 5B, “Average”). In contrast, low temperature (23°C), the only condition that decreased the rate of recombination, lead to a modest increase in the fraction. Together, these observations suggest an association between increased recombination activity and loss of IC/IS bias. Finally, results also showed that the effects of different conditions on IC/IS bias can be either RSZ specific, as in the case of HU, or general, as in the case of temperature, rrm3Δ, and mec1Δ sml1Δ (Figure 5B legend).
We found that rates of URA3 inactivation at RSZs were comparable to non-RSZ loci under all tested conditions. Based on this, we conclude that RSZs are not recombination hotspots, defined as loci of increased recombination activity. A key assumption is that the URA3 inactivation event monitored in the current study is a readout for recombination activities at the locus, and not for an indirect effect (s) of a more distant element (s). For example, studies of LOH in diploids indicate that most LOH events occur by a crossover between the heterozygous loci, and that the frequency of these events increases as a function of distance from centromere (e. g. [31]). In addition, Ty elements are recombination hotspots that might affect recombination activities downstream of the loci (e. g. [32]). Notably however, the frequency of the LOH events monitored in the current study was independent of the distance from CENIII (Figure 3I, J). Similarly, the rates of overall URA3 inactivation at the loci downstream (the three non RSZ loci) and upstream (the two RSZ loci) of a Ty element were comparable (Figure 2B, 4B, and 5A). Furthermore, the approach employed in the current study – i. e. integration of a reporter construct at a specific locus - is a widely utilized means of assessing local recombination activities (e. g. [33]). Together, these considerations strongly suggest that the assay utilized in the current study monitors local recombination activity. It was surprising that RSZs did not have intrinsically higher rates of recombination than non RSZs. However, the current observation is actually consistent with the fact that RSZ breakage occurs during prometaphase in the context of topoisomerase II- and condensin- dependent chromosome condensation [3], [20], while most spontaneous mitotic recombination occurs during S phase in the context of stalled- and collapsed-replication forks [5], [7], [26]. The apparent temporal separation and differential genetic requirements suggest that chromosome breakage and recombination at RSZs might each entail a process that is independently regulated. However, it is also possible that the lack of correlation is a feature specific to RSZ (and mammalian fragile sites, by extension) and not a general feature of a fragile site. Our results confirmed an earlier observation that the locus-to-locus variation in spontaneous mitotic recombination rate is relatively modest, in contrast to the variation in meiotic recombination rates, which can be several orders of magnitude [34], [35]. This difference is likely due, at least in part, to the nature of DNA structure that leads to recombination in each case: stalled- or collapsed-replication forks for mitotic recombination and developmentally programmed DSBs for meiotic recombination [26], [36]. Formation of meiotic DSBs are regulated at multiple levels, including targeted localization of Spo11, the enzyme that catalyzes the breakage, to the hotspot regions of the genome [37]. This in turn ensures that meiotic DSBs do not occur uniformly throughout the genome, but preferentially at DSB hotspots. In contrast, the occurrence of stalled- or collapsed-forks leading to spontaneous mitotic recombination does not appear to be regulated per se; rather, it appears to be a unintended consequence of a replication fork encountering a locus that is difficult to replicate, for example, due to either unusual DNA or chromatin structures or damaged DNA [6], [7], [25], [26], [38]. Previous studies have shown that genes encoding for proteins involved in processes such as replication fork progression, stalled fork integrity, and fork restart impact endogenous mitotic recombination rates (e. g. [39], [40]). The recombinogenic effects of HU or rrm3Δ observed in current study are likely due to increased incidences (either in the frequency and/or the duration) of fork stalling stemming from depletion of dNTP pools or loss of a replisome associated helicase activity, respectively [28], [41]. The effects of mec1Δ sml1Δ, on the other hand, is unlikely due to increased fork stalling because replication forks proceed faster in an sml1Δ background compared to WT [3]. Given that Mec1 is required for stability of stalled forks and is a key regulator of homologous recombination and recombination related processes involved in replication fork restart [18], [42], the recomginogenic effects of mec1Δ sml1Δ are likely to stem from a defect (s) in processes that occur after fork stalling. The mechanism (s) by which temperature might affect endogenous recombination activity remains unknown. Notably however, there has been a precedent in meiotic recombination, where temperature appears to play an important regulatory role (s) [43]. On average, URA3 inactivation under the standard growth condition was four times more likely to occur via an IC/IS mediated event than all other mechanisms combined. We found that HU, high temperature, rrm3Δ, and mec1Δ sml1Δ abolished this bias while low temperature and sml1Δ enhanced it. These observations suggest that partner choice in mitotic recombination might be subjected to regulation, for example by factors like dNTP availability and temperature. During meiotic recombiantion, a significant fraction of meiotic DSBs is developmentally programmed to be repaired with an IH bias, using an intact homolog as a repair template, rather than a sister chromatid. Evidence indicates that such IH bias in meiotic recombination is mediated, at least in part, by expression of several meiosis specific chromosomal proteins that fundamentally alter meiotic chromatin structure; this in turn, favours physical interaction between the homologs while minimizing that between sister chromatids, overcoming the IC/IS bias intrinsic during mitotic recombination [44], [45], [46]. The latter implicates chromatin structure, notably the status of sister chromatid cohesion, in mitotic IC/IS bias. We utilized RSZ, a model for mammalian common fragile sites, to address the proposal that the fragile sites contribute to cancer due to increased recombination activity at the loci. The evidence presented here, however, suggests an alternative mechanism, which implicates the nature of recombination outcomes, rather than the overall recombination rate. The only RSZ specific recombination activity revealed in the current study was its greater sensitivity to HU induced loss of IC/IS bias. Although the sample size is limiting (i. e. one insertion in each of two RSZs), an implication would be that, depending on the nature of stress, recombination events at RSZs (and at mammalian common fragile sites, by extension), might be more likely to lead to a LOH and translocation, the type of alterations shown to contribute to cancer. Taken together, current observations provide a fresh insight into the ways in which fragile sites and other recombinogenic conditions may contribute to genome rearrangements.
Relevant genotypes of the strains utilized in current study are summarized in Table S1. All URA3 strains were generated by standard yeast genetics procedures including transformation, mating, sporulation, and specific phenotype selections. A wild type haploid strain NKY291, was transformed with each of the five DNA fragments containing the hisG-URA3-hisG cassette flanked by ∼500 bp upstream (L) and ∼500 bp downstream (R) genomic sequences of the targeted loci. The fragments were prepared from integration plasmids (Figure S1) by NotI digestion and gel purification. Correct integration of hisG-URA3-hisG at each locus among randomly selected URA transformants was confirmed by Southern Blot analysis (Figure S3). Two independent transformants of each locus were mated with NKY292, a WT haploid strain of the opposite mating type to generate heterozygous diploid strains, from which URA3 haploids were re-derived. These haploid strains (JDCY 463,465,230,233,239,243,232,233,235,237) were used for all subsequent strain construction. The rate of URA3 inactivation was determined by the method of median [24]. For each measurement, 15 colonies of comparable size (1. 5–2 mm in diameter) freshly grown on YPD plates, were individually suspended in 5 mls of YPD or YPD+10 mM HU and cultured for 18 hours at 30°C (Figure S2A). Samples were then diluted in water and plated on YPD or 5FOA plates to measure the number of total viable cells or those that had undergone a URA3 inactivation event, respectively. To ensure that only the URA3 inactivation events that occurred during the 18 hour of unselected growth in liquid medium were included for the analyses, only the 5FOARcolonies of comparable size (1. 5–2 mm) were counted following a three day incubation at 30°C. Statistical analyses on the rates of URA3 inactivation were assessed as described [47]. For each condition examined, a total of 20 or more independent 5FOAR colonies from each locus were subjected to molecular analysis (Figure S2B). Genomic DNA from each colony was restricted using the appropriate restriction enzyme (Figure S3; Figure 3) and subjected to Southern Blot analysis. For each locus, the L and R fragments used for plasmid construction (Figure S1) were used as probes. As a control for quantifying the relative signals associated with different diagnostic fragments (e. g. Figure 3E), a PCR amplified Mec1 fragment corresponding to nucleotide numbers 5539 to 7027 in the OFR was included as a probe (Table S2). Each restriction enzyme used for Southern analysis cleaves endogenous MEC1 sequence to generate a novel sized fragment that hybridizes to the MEC1 probe. To confirm the occurrence of ectopic recombination events, the candidate 5FOAR colonies were analyzed by Pulse Field Gel electrophoresis (PFGE) and Southern Blot analyses using hisG as a probe. For PFGE, Chromosome sized genomic DNA samples were prepared in low melting point agarous plugs as previously described [48]. Electrophoresis condition optimized for resolution around ChrIII was performed as described [49].
Chromosome rearrangements are frequently associated with human cancers. Such rearrangement can result from a DNA break followed by an erroneous repair. Mammalian common fragile sites are one of the most extensively studied naturally occurring breakage prone regions of the genome. It has been proposed that fragile sites are recombination hotspots and that increased recombination activity at these loci contribute to cancer. We examined this hypothesis using a model organism, budding yeast Saccharomyces cerevisiae, where a homolog of the mammalian common fragile sites has been identified. Unexpectedly, our results showed that the rate of recombination at the fragile sites was not any higher than non fragile sites, even under the conditions that promoted chromosome breakage at the fragile sites. However, we found that the frequency of loss of heterozygosity (LOH) and translocation, the type of recombination outcomes known to contribute to cancer, to be significantly elevated at fragile sites under certain conditions. These findings suggest that the fragile sites might indeed contribute to cancer more frequently than non-fragile loci, but the reason for this is likely to be due the nature of the recombination outcome (s) rather than higher rates of recombination.
Recombinogenic Conditions Influence Partner Choice in Spontaneous Mitotic Recombination