Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination.     

Break induced replication in eukaryotes: mechanisms,functions, and consequences

Break-induced replication (BIR) is an important pathway specializing in repair of one-ended double-strand DNA breaks (DSBs). This type of DSB break typically arises at collapsed replication forks or at eroded telomeres. BIR initiates by invasion of a broken DNA end into a homologous template followed by initiation of DNA synthesis that can proceed for hundreds of kilobases. This synthesis is drastically different from S-phase replication in that instead of a replication fork, BIR proceeds via a migrating bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual mode of DNA replication is responsible for frequent genetic instabilities associated with BIR, including hyper-mutagenesis, which can lead to the formation of mutation clusters, extensive loss of heterozygosity, chromosomal translocations, copy-number variations and complex genomic rearrangements. In addition to budding yeast experimental systems that were initially employed to investigate eukaryotic BIR, recent studies in different organisms including humans, have provided multiple examples of BIR initiated within different cellular contexts, including collapsed replication fork and telomere maintenance in the absence of telomerase. In addition, significant progress has been made towards understanding microhomology-mediated BIR (MMBIR) that can promote complex chromosomal rearrangements, including those associated with cancer and those leading to a number of neurological disorders in humans.     

Break-Induced DNA Replication

Recombination-dependent DNA replication, often called break-induced replication (BIR), was initially invoked to explain recombination events in bacteriophage but it has recently been recognized as a fundamentally important mechanism to repair double-strand chromosome breaks in eukaryotes. This mechanism appears to be critically important in the restarting of stalled and broken replication forks and in maintaining the integrity of eroded telomeres. Although BIR helps preserve genome integrity during replication, it also promotes genome instability by the production of loss of heterozygosity and the formation of nonreciprocal translocations, as well as in the generation of complex chromosomal rearrangements.The break-copy mode of recombination (as opposed to break-join), was initially proposed by Meselson and Weigle (1961). Break-copy recombination, now more commonly known as recombination-dependent DNA replication or break-induced replication (BIR), is believed to account for restarting replication at broken replication forks and may also play a central role in the maintenance of telomeres in the absence of telomerase. BIR has been studied in various model systems and has been invoked to explain chromosome rearrangements in humans. This review focuses primarily on mechanistic studies in Escherichia coli and its bacteriophages, T4 and λ, in the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and on apparently similar, but less well-documented, mechanisms in mammalian cells.Homology-dependent repair of DNA double-strand breaks (DSBs) occur by three major repair pathways (Pâques and Haber 1999) (Fig. 1). When both ends of the DNA share substantial homology with a donor template (a sister chromatid, a homologous chromosome, or an ectopically located segment), repair occurs almost exclusively by gene conversion (GC). If the DSB is flanked by direct repeats, then a second repair process, single-strand annealing (SSA), can occur as 5′ to 3′ resection of the DSB ends exposes complementary sequences that can anneal to each other and repair the break by the formation of a deletion. However, when only one DSB end shares homology with a donor sequence, repair occurs by BIR. There are two BIR pathways, one dependent on Rad51 recombinase and the other independent of Rad51.Open in a separate windowFigure 1.Three major repair pathways of homology-dependent recombination. Noncrossover (NCO) and crossover (CO) events are indicated. Black triangles represent resolution of Holliday junctions (HJs). Dashed lines represent new DNA synthesis. GC, gene conversion; SSA, single-strand annealing; BIR, break-induced replication.     

Loss of heterozygosity is induced in <Emphasis Type="Italic">Candida albicans</Emphasis> by ultraviolet irradiation

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.     

Neocentromeres Form Efficiently at Multiple Possible Loci in Candida albicans

Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-A^Cse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.     

Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.     

Aberrant Double-Strand Break Repair Resulting in Half Crossovers in Mutants Defective for Rad51 or the DNA Polymerase δ Complex

Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Δ mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Δ and pol3-ct mutants, defective for components of the DNA polymerase δ (Pol δ) complex. The half crossovers formed in Pol δ complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Δ mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol δ complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.DNA double-strand breaks (DSBs) are potentially lethal lesions that can occur spontaneously during normal cell metabolism, by treatment of cells with DNA-damaging agents, or during programmed recombination processes (54). There are two major pathways to repair DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves the religation of the two ends of the broken chromosome and can occur with high fidelity or be accompanied by a gain or loss of nucleotides at the junction (9). Repair of two-ended DSBs by HR generally occurs by gene conversion resulting from a transfer of information from the intact donor duplex to the broken chromosome (Fig. ​(Fig.1).1). HR occurs preferentially during S and G₂ when a sister chromatid is available to template repair (2, 19, 22). Sister-chromatid recombination events are genetically silent, whereas gene conversion between nonsister chromatids associated with an exchange of flanking markers can result in extensive loss of heterozygosity (LOH) or chromosome rearrangements (3, 21). One-ended DSBs that arise by replication fork collapse or by erosion of uncapped telomeres are thought to repair by strand invasion into homologous duplex DNA followed by replication to the end of the chromosome, a process referred to as break-induced replication (BIR) (35). BIR appears to be suppressed at two-ended breaks, presumably because it can lead to extensive LOH if it occurs between homologues or to chromosome translocations when strand invasion initiates within dispersed repeated sequences (5, 28, 31, 50, 52, 55).Open in a separate windowFIG. 1.Models for gene conversion and BIR. After formation of a DSB, the ends are resected to generate 3′ single-strand DNA tails. One end undergoes Rad51-dependent strand invasion to prime DNA synthesis from the invading 3′ end templated by the donor duplex. For gene conversion by the synthesis-dependent strand annealing model, the extended invading end is displaced and can anneal to the other side of the break; completion of repair requires DNA synthesis primed from the noninvading 3′ end. For a one-ended break, or if the other side of the break lacks homology to the donor duplex, DNA synthesis proceeds to the end of the chromosome. Centromeres are shown as solid ovals and a heterozygous marker centromere distal to the site of repair as A/a.The strand invasion step of BIR is assumed to be the same as that for gene conversion based on the requirement for the same HR proteins: Rad51, Rad52, Rad54, Rad55, and Rad57 (10). However, subsequent steps in BIR are less well defined. Recent studies of the fate of the invading end during BIR in diploid strains with polymorphic chromosome III homologues using a plasmid-based assay have shown that following strand invasion, the invading end is capable of dissociating from the initial homologous template. Following dissociation, the displaced end subsequently reinvades into the same or a different chromosome III homologue by a process termed template switching (52). One of the interesting features of the template switching events is that they occur over a region of about 10 kb downstream of the site of strand invasion and do not extend over the entire left arm of chromosome III. There are a number of possible mechanisms that could account for this apparent change in the processivity of BIR. First, it is possible that the strand invasion intermediate is cleaved by a structure-specific nuclease and once the invading strand is covalently joined to one of the template strands, the strand invasion process is irreversible. Recent studies of Schizosaccharomyces pombe have shown an essential role for Mus81, a structure-specific nuclease, in resolution of sister chromatid recombination intermediates during repair of collapsed replication forks (48). Another possibility is that there could be a switch between a translesion DNA polymerase and a highly processive DNA polymerase during BIR. The translesion polymerases in budding yeast, polymerase ζ (Pol ζ) and Pol η, are encoded by REV3-REV7 and RAD30, respectively (34, 40, 43). Deletion of REV3 has been shown to increase the fidelity of DNA synthesis associated with HR but has no effect on the overall frequency of DSB-induced HR (16). Deletion of POLη in chicken DT40 cells reduces the frequency of DSB-induced gene conversion, and human POL η has been shown to extend the invading 3′ end of D-loop intermediates in vitro (23, 36). However, this same preference for Pol η is not found for Saccharomyces cerevisiae. Instead, DNA synthesis during meiotic and mitotic recombination appears to be carried out by Pol δ, one of the three nuclear replicative polymerases, which normally functions with Pol α in Okazaki fragment synthesis (13, 32, 33, 44). Pol ɛ is thought to be the primary leading-strand polymerase (47), but in the absence of the Pol ɛ catalytic domain, Pol δ is presumed to carry out leading-strand synthesis (24). Recent studies by Lydeard et al. (30) have shown a requirement for the lagging-strand polymerases, Pol δ and Pol α, to form the initial primer extension product during BIR, and Pol ɛ is required to complete replication to the end of the chromosome. In contrast, repair of DSBs by gene conversion does not require Pol α, and there appears to be functional redundancy between Pol δ and Pol ɛ (56).To address the roles of Mus81, Pol δ, and Pol η in BIR and in particular template switching, we used the transformation-based BIR assay with diploids with polymorphic chromosome III homologues. Because the transformation assay can only be used with strains with viable mutations of replication factors, we used a null allele of POL32, encoding a nonessential subunit of the Pol δ complex (14), and a point mutation in the gene encoding the essential catalytic subunit, POL3. The pol3-ct allele results in a truncation removing the last four amino acids of the Pol3 protein; the C-terminal region of Pol3 is implicated in interaction with the other essential subunit of the Pol δ complex, Pol31 (15, 49). The interesting feature of the pol3-ct allele is that it decreases the length of gene conversion tracts during mitotic and meiotic recombination, presumably by affecting the processivity of Pol δ, but confers no apparent defect in normal DNA synthesis (32, 33). Because BIR requires more-extensive tracts of DNA synthesis than gene conversion, we expected the pol3-ct mutant to exhibit a BIR defect. We found that in the absence of a fully functional Pol δ complex, chromosome fragment (CF) formation proceeds by a half-crossover mechanism associated with loss of the template chromosome, an event with potentially catastrophic consequences (6, 57). This was also found to occur in rad51 mutants, suggesting nonreciprocal translocations arise by failure to undergo strand invasion or because replication following strand invasion is inefficient. In contrast to rad51 mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion and fully resistant to ionizing radiation, suggesting there is a unique requirement for Pol δ to complete BIR. Consistent with studies of gene conversion in S. cerevisiae (33), we found no role for Pol η in BIR or the process of template switching.     

Break-induced replication is highly inaccurate

DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.     

RecA protein assures fidelity of DNA repair and genome stability in Deinococcus radiodurans

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5 kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5 kGy γ-radiation, extremely high γ-doses, e.g., 25 kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.     

Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.     

Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome.

A method was devised for extracting, from cells of Escherichia coli K12, DNA molecules which sedimented on neutral sucrose gradients as would be expected for free DNA molecules approaching the genome in size. Gamma ray irradiation of oxygenated cells produced 0.20 DNA double-strand breaks per kilorad per 10⁹ daltons. Incubation after irradiation of cells grown in K medium, with four to five genomes per cell, showed repair of the double-strand breaks. No repair of double-strand breaks was found in cells grown in aspartate medium, with only 1.3 genomes per cell, although DNA single-strand breaks were still efficiently repaired. Cells which were recA^? or recA^?recB^? also did not repair double-strand breaks. These results suggest that repair of DNA double-strand breaks may occur by a recombinational event involving another DNA double helix with the same base sequence.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

High-resolution genome-wide analysis of irradiated (UV and γ-rays) diploid yeast cells reveals a high frequency of genomic loss of heterozygosity (LOH) events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.     

Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr⁵⁴⁰ amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.     

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.     

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A^CaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A^CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A^CaCse4 levels at the programmed stall sites of early replicating centromeres.