Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila

In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis

Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition

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

DNA Resection at Chromosome Breaks Promotes Genome Stability by Constraining Non-Allelic Homologous Recombination

DNA double-strand breaks impact genome stability by triggering many of the large-scale genome rearrangements associated with evolution and cancer. One of the first steps in repairing this damage is 5′→3′ resection beginning at the break site. Recently, tools have become available to study the consequences of not extensively resecting double-strand breaks. Here we examine the role of Sgs1- and Exo1-dependent resection on genome stability using a non-selective assay that we previously developed using diploid yeast. We find that Saccharomyces cerevisiae lacking Sgs1 and Exo1 retains a very efficient repair process that is highly mutagenic to genome structure. Specifically, 51% of cells lacking Sgs1 and Exo1 repair a double-strand break using repetitive sequences 12–48 kb distal from the initial break site, thereby generating a genome rearrangement. These Sgs1- and Exo1-independent rearrangements depend partially upon a Rad51-mediated homologous recombination pathway. Furthermore, without resection a robust cell cycle arrest is not activated, allowing a cell with a single double-strand break to divide before repair, potentially yielding multiple progeny each with a different rearrangement. This profusion of rearranged genomes suggests that cells tolerate any dangers associated with extensive resection to inhibit mutagenic pathways such as break-distal recombination. The activation of break-distal recipient repeats and amplification of broken chromosomes when resection is limited raise the possibility that genome regions that are difficult to resect may be hotspots for rearrangements. These results may also explain why mutations in resection machinery are associated with cancer

A Microhomology-Mediated Break-Induced Replication Model for the Origin of Human Copy Number Variation

Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV). A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2–5 base pairs (bp). Third, endpoints occur near pre-existing low copy repeats (LCRs). Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR) for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR). Under these circumstances, single-strand 3′ tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication

Formation of Complex and Unstable Chromosomal Translocations in Yeast

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers

Identification of Trypanosoma brucei RMI1/BLAP75 Homologue and Its Roles in Antigenic Variation

At any time, each cell of the protozoan parasite Trypanosoma brucei expresses a single species of its major antigenic protein, the variant surface glycoprotein (VSG), from a repertoire of >2,000 VSG genes and pseudogenes. The potential to express different VSGs by transcription and recombination allows the parasite to escape the antibody-mediated host immune response, a mechanism known as antigenic variation. The active VSG is transcribed from a sub-telomeric polycistronic unit called the expression site (ES), whose promoter is 40–60 kb upstream of the VSG. While the mechanisms that initiate recombination remain unclear, the resolution phase of these reactions results in the recombinational replacement of the expressed VSG with a donor from one of three distinct chromosomal locations; sub-telomeric loci on the 11 essential chromosomes, on minichromosomes, or at telomere-distal loci. Depending on the type of recombinational replacement (single or double crossover, duplicative gene conversion, etc), several DNA-repair pathways have been thought to play a role. Here we show that VSG recombination relies on at least two distinct DNA-repair pathways, one of which requires RMI1-TOPO3α to suppress recombination and one that is dependent on RAD51 and RMI1. These genetic interactions suggest that both RAD51-dependent and RAD51-independent recombination pathways operate in antigenic switching and that trypanosomes differentially utilize recombination factors for VSG switching, depending on currently unknown parameters within the ES

Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions

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

The DNA Damage Response Pathway Contributes to the Stability of Chromosome III Derivatives Lacking Efficient Replicators

In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1AQ allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps