Biology subjects

Haber, J. E.

Publications and source records attributed to Haber, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Replication Fork-Associated Checkpoint Mediator Mrc1/Claspin Acts in Arrest-Independent Double-Strand Break Survival

In budding yeast, the DNA damage checkpoint kinase Mec1ATR is the primary sensor of DSB-associated single-stranded DNA and coordinates cell cycle arrest with DNA repair. Deletion of Mec1 completely abolishes G2/M cell cycle arrest; however, approximately half of mec1{Delta} cells still survive an endonuclease-induced DSB, repairing the DSB either by single-strand annealing or break-induced replication. Here we show that Mec1-independent DSB repair is independent of Rad9 but requires the Tel1ATM kinase, Rad53 kinase, and the 9-1-1 sliding clamp, which promotes Tel1 retention at damage sites. We identify Mrc1Claspin, a replication fork-associated checkpoint mediator, as an unexpected contributor to DSB survival in the absence of Mec1. Unlike Rad9, the canonical DNA damage adaptor, Mrc1's role in DSB survival is independent of its Mec1/Tel1 consensus phosphorylation sites and relies primarily on its C-terminal domain. Mrc1 accumulates at a single DSB and both promotes end-tethering and limits DNA end-resection in an S-phase-specific manner. Mrc1 functions independently of its replication checkpoint partners, Tof1 and Csm3. We further show that heterochromatic gene silencing in budding yeast is Mrc1-dependent but Tof1- and Csm3-independent. Together, these findings define a Mec1-independent survival pathway and establish Mrc1 as a novel regulator of DSB repair.

molecular biology↗

bioRxiv2025

Esc1-mediated anchoring regulates telomere clustering in response to metabolic changes.

Spatial organization of yeast telomeres is highly dynamic and regulated by growth conditions. In rich medium, the 32 telomeres group in 3 to 5 foci at the nuclear periphery. This organization is drastically rearranged in long-lived quiescent (Q) cells forming upon carbon source exhaustion: telomere foci assemble into a hypercluster containing most telomeres and located in the center of the nucleus contributing to their long-term viability. Here we explore the mechanisms regulating telomere distribution during this transition. We rule out a modification of telomere-telomere interactions via the telomeric protein Sir3 as the main factor regulating hypercluster formation. However, our physical modeling predicts that telomere anchoring antagonizes telomere clustering. Systematic deletion of all known telomere anchors identifies the inner nuclear membrane-associated protein Esc1, as a key telomeric anchor after the diauxic shift. Our data support a model where Esc1 mediated anchoring is progressively lost upon entry into quiescence through dephosphorylation of a single residue of Esc1, resulting in the formation of telomere hyperclusters in the center of the nucleus in Q cells.

cell biology↗

bioRxiv2025

Mutations and structural variants arising during double-strand break repair

Double-strand break (DSB) repair is highly mutagenic compared to normal replication. In budding yeast, repair of an HO endonuclease-induced DSB at MAT can be repaired by using a transcriptionally silent HMR::Kl-URA3 donor. During repair, -1 deletions in homonucleotide runs are strongly favored over +1 insertions, whereas during replication, spontaneous +1 and -1 events are equal. Microhomology-bounded, repair-associated intragenic deletions (IDs) are recovered 12 times more frequently than tandem duplications (TDs). IDs have a mean length of 56 bp, while TDs average 22 bp. These data suggest a picture of the structure of the repair replication fork: IDs and TDs occur within the open structure of a migrating D-loop, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either [~]80 bp ahead or [~]40 bp behind the 3 end. Another major class of repair-associated mutations ([~]10%) are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence in HMR is only 72% identical (homeologous) with S. cerevisiae ura3-52. ICTS events begin and end at regions of short ([~]7 bp) microhomology; however, ICTS events are constrained to the middle of the copied sequence. Whereas microhomology usage in intragenic deletions is not influenced by adjacent homeology, we show that extensive pairing of adjacent homeology plays a critical role in ICTS. Thus, although by convention, structural variants are characterized by the precise base pairs at their junction, microhomology-mediated template switching actually requires alignment of extensive adjacent homeology. Significance statementDNA synthesis during repair of a double-strand chromosome break by homologous recombination exhibits a high rate of mutation compared to normal replication. Using a genetic system in budding yeast, we isolated thousands of mutations occurring during repair. We conclude that the repair replication fork appears to have the two DNA strands open [~]80 bp ahead of the DNA polymerase, but the strands re-anneal rapidly behind the polymerase. Additionally, we analyzed interchromosomal template switching, in which the partially copied DNA strand dissociates and pairs with a new template at a short stretch of perfectly matching bases (microhomology), and resumes copying. We show that these apparent microhomology-mediated template switching events in fact require the pairing of [~]200 bp of imperfectly matching bases (homeology).

genetics↗

bioRxiv2025

Yeast DNA Polymerase δ Proofreading Nuclease Suppresses Translesion Synthesis and Facilitates Recombination in DNA Lesion Bypass.

Translesion synthesis (TLS) employs specialized polymerases to incorporate nucleotides opposite DNA lesions, allowing replication to continue temporarily without repairing the lesions, while sacrificing replication fidelity (1). TLS depends on a ubiquitin molecule attached to the clamp PCNA at residue Lys164 (2, 3); the precise biochemical and molecular basis how PCNA modification stimulates TLS is still elusive (4-10). In this study, we discovered that DNA polymerase {delta} (Pol {delta}) 3 to 5 "proofreading" exonuclease degrades nascent TLS synthesis opposite pyrimidine dimers and inhibits TLS on both leading and lagging strands. Rad18 E3 ligase and PCNA ubiquitylation become dispensable for TLS when Pol {delta} exonuclease is inactive. Biochemical reconstitution showed that the major UV lesion bypass polymerase Rad30 is intrinsically inefficient at bypassing thymidine dimers and requires multiple turnovers. Pol {delta} exonuclease impedes Rad30 activity both catalytically and non-catalytically during and post-lesion bypass. Importantly, PCNA and PCNA ubiquitylation enhance processivity of Rad30 after thymidine dimer bypass, a critical step for further DNA synthesis extension and overcoming Pol {delta} inhibition. Our study reveals a new paradigm how PCNA ubiquitylation facilitates TLS and the new role of Pol {delta} nuclease in lesion bypass pathway choice and replication fidelity at fork-blocking DNA lesions.

genetics↗

bioRxiv2024

Dissecting quantitative trait nucleotides by saturation genome editing

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.

genetics↗

bioRxiv2023

Detecting significantly recurrent genomic connections from simple and complex rearrangements in the cancer genome

The detection of somatic genetic alterations that recur across cancer genomes more than expected by chance has been a major goal of cancer genomics, as these alterations are enriched for "driver" events that promote cancer. Multiple methods have been developed to detect driver point mutations and copy-number variants, but methods to detect driver rearrangements have largely not been pursued. Unlike point mutations and copy-number alterations, which can be assigned to a single genomic locus, rearrangements connect two distant genomic loci, and possibly more in the case of complex or clustered events. Here, we explore genomic features that predict the rate at which any pair of loci will be connected by rearrangements and describe two methods to detect rearrangements that recur more often than this background rate. The first, SVSig-2D, detects pairs of loci that are directly connected by a single rearrangement; the second, SVSig-2Dc also detects loci that are recurrently connected indirectly through two or more rearrangements. When applied to a pan-cancer dataset of over 2,500 cancers, these methods identified 80 significantly recurrent simple rearrangements and 29 complex rearrangements, including both known and novel events. Intriguingly, though both recurrent simple and complex rearrangements tended to be tissue-specific, this was less true for the complex events. The detection of recurrent rearrangements with methods such as these will be an essential component of cancer genomics in the whole-genome sequencing era.

genomics↗

bioRxiv2023

Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork

Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MAT locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3- mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are -1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and -1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase {delta} copies the template, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3 end, within the open structure of a migrating D-loop. Our data suggest that [~]100 bp ahead of the polymerase is "open," but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.

genetics↗

bioRxiv2023

Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially-copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MAT, repaired by an HMR::Kl-URA3 donor. We inserted into HMR::Kl-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield mat::Kl-ura3::QP+4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP+4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP+4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (pol3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a +1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura3+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

genetics↗

bioRxiv2023

Prolonged DNA Damage Checkpoint Arrest Requires Constant Renewal and the Spindle Assembly Checkpoint

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

genetics↗

bioRxiv2023

The Kinase Module of the Mediator regulates cell cycle re-entry and transcriptional response following DNA damage

The Cdk8 kinase module (CKM) is a non-obligate and dissociable subcomplex of Mediator of transcription, a key regulator of RNA polymerase II (RNAPII). Through a genetic screen in yeast, we discovered a surprising role for Mediator CKM in the DNA damage response (DDR) and mitotic re-entry. Remarkably, we find that a single DNA break is sufficient for CKM-dependent global transcriptional attenuation. Upon DDR activation, the kinase activity of CKM antagonizes RNAPII binding to core Mediator, thereby reducing the transcriptionally-engaged RNAPII pool. This transcriptional attenuation is essential for DDR inactivation and limits the spreading of {psi}-H2AX into gene bodies. Furthermore, CKM localizes to DNA breaks to impede RNAPII binding. Importantly, we demonstrate that the role of CKM on DDR and transcriptional attenuation is conserved from yeast to mammals, establishing a multifaceted and essential function for CKM in transcriptional regulation of DNA-damage response.

cell biology↗

bioRxiv2022

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.3-kb of phage lambda DNA in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3 and Slx4. A natural 3% divergence between repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL) are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the identical repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain, where the F variant carries six single-base mutations and one +1(T) insertion within a run of 10 Ts showed a gradient of correction favoring the sequence opposite each 3 end of the annealed strand. Mismatches (MMs) located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, using either 3 nonhomologous tail removal or 3 to 5 "proofreading" resection by DNA polymerase {delta} followed by synthesis to fill in the gap. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat, with a bias in favor of the "left" copy. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, while the deletion of MSH6 resulted in unrepaired MMs.

genetics↗

bioRxiv2022

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

microbiology↗

bioRxiv2022

Rad51 and Dmc1 have similar tolerance for mismatches in yeast meiosis

In many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51s homolog, Dmc1, acting with Rad54s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease, either under control of a galactose-inducible promoter or a meiosis-specific SPO13 promoter. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which a 108-bp homologous sequence shared between the DSB end and the donor sequence could be easily modified. As previously shown for a haploid mitotic strain, BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic haploids or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Rad51 took over from Dmc1 in both hed1{Delta} and dmc1{Delta} hed1{Delta} mutants. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination in budding yeast. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. In all cases, assimilation of mismatches into the BIR product was dependent on the 3 to 5 exonuclease activity of DNA polymerase {delta}. Author SummaryIn many eukaryotes, including both budding yeast and mammals, repair of double-strand breaks (DSBs) is carried out by different apparatus in somatic and meiotic cells. In mitotic cells, Rad51 recombinase, acting with Rad54, facilitates the search for homology and DNA strand exchange. In budding yeast meiosis, Rad51 is inhibited by Hed1 and plays only an effector role, while strand exchange is driven by Rad51s homolog, Dmc1, acting with Rad54s homolog, Rdh54/Tid1. To directly compare the activities of Rad51 and Dmc1 and especially their tolerance for recombination between divergent sequences, we created diploids in which a site-specific DSB was created by HO endonuclease. Homologous recombination was measured by an ectopic break-induced replication (BIR) assay in which recombination occurred between a 108-bp homologous sequence shared between the DSB end and the donor sequence. The donor sequence could be easily modified to introduce different arrangements of mismatches. BIR efficiency decreased with increasing divergence between donor and recipient, but repair occurred even when every 6th base pair was mismatched. There was little difference in the tolerance of mismatches in mitotic or meiotic diploids; however, there were notable differences in meiotic diploids when recombination was facilitated by Dmc1 or when Dmc1 was deleted and Rad51 was activated. We found that Dmc1 and Rad51 are similarly tolerant of mismatches during meiotic recombination. Surveillance of mismatches by the Msh2 mismatch repair protein proved to be Dmc1-specific. As in mitotic cells, the assimilation of mismatches into the BIR product was dependent on the 3 to 5 exonuclease activity of DNA polymerase {delta}.

genetics↗

bioRxiv2022

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks

Expanded CAG/CTG repeats are sites of DNA damage, leading to changes in repeat length. To determine how ssDNA gap filling affects repeat instability, we inserted (CAG)70 or (CTG)70 repeats into a single-strand annealing (SSA) assay system such that resection and filling in the ssDNA gap would occur across the repeat tract. After resection, when the CTG sequence was the single-stranded template for fill-in synthesis, repeat contractions were elevated and the ssDNA created a fragile site that led to large deletions involving flanking homologous sequences. In contrast, resection was inhibited when CTG was on the resected strand, resulting in repeat expansions. Deleting Rad9, the ortholog of 53BP1, rescued repeat instability and lost viability by increasing resection and fill-in speed. Deletion of Rad51 increased CTG contractions and decreased survival, implicating Rad51 in protecting ssDNA during gap filling. Taken together, DNA sequence within a single-stranded gap determines repair kinetics, fragility, and repeat instability.

genetics↗

bioRxiv2022

Repair of Mismatched Templates during Rad51-dependent Break-Induced Replication

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3 end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. When every 10th base is mismatched between donor and recipient, BIR is 44% efficient compared to fully homologous sequences; but BIR still occurs about 10% when every 6th base is mismatched. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, but where they are clustered with spacings of every 6th bp. These different arrangements of uneven mismatch distribution were in general less efficient in recombination as templates with evenly distributed mismatches. A donor with all 10 mismatches clustered every 6th base at the 3 invading end of the DSB was not impaired compared to the case where mismatches were clustered at the 5 end. These data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but sequence-specific factors are also important. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3 end. Mismatch incorporation depends on the 3 [->] 5 proofreading exonuclease activity of DNA polymerase {delta}, with little contribution from Msh2/Mlh1 mismatch repair proteins. Surprisingly, the probability of a mismatch 27 nt from the 3 end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase {delta} "chews back" the 3 end of the invading strand without any mismatch-dependent cues from the strand invasion structure. Author SummaryDNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3 end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template and found that BIR efficiency was the same whether the mismatches were clustered at the 3 invading end or at the 5 end. We confirmed that mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3 end. We conclude that the proofreading activity of DNA polymerase {delta} "chews back" the 3 end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. These observations enrich our understanding of the details of Rad51-mediated strand invasion and provide insight into the mechanism of the 3 to 5 proofreading activity of DNA polymerase during homologous recombination.

molecular biology↗

bioRxiv2021

Euchromatin factors HULC and Set1C affect heterochromatin organization for mating-type switching in fission yeast Schizosaccharomyces pombe

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1{Delta}. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

genetics↗

bioRxiv2020

Chromosome structure due to phospho-mimetic H2A modulates DDR through increased chromatin mobility

In budding yeast and mammals, double strand breaks trigger global chromatin mobility together with the rapid phosphorylation of the histone H2A over an extensive region of the chromatin. To assess the role of H2A phosphorylation in this response to DNA damage, we have constructed strains where H2A has been mutated to the phospho-mimetic H2A-S129E. We show that H2A-S129E mutant increases global motion of chromosomes even in the absence of DNA damage. The intrinsic chromatin mobility of H2A-S129E is not due to checkpoint activation, histone degradation or kinetochore anchoring. Rather, the increased intra-chromosomal distances observed in H2A-S129E mutant are consistent with chromatin structural changes. In this context, the Rad953BP1-dependent-checkpoint becomes dispensable. The increase in chromatin dynamics is favorable to NHEJ of a single double-strand break but is accompanied by a sharp decrease in inter-chromosomal translocation rates. We propose that changes in chromosomal conformation due to H2A phosphorylation are sufficient to modulate the DDR and maintain genome integrity.

genetics↗

bioRxiv2020

Mechanisms preventing Break-Induced Replication during repair of two-ended DNA double-strand breaks

DNA synthesis during homologous recombination (HR) is highly mutagenic and prone to template switches. Two-ended DNA double strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by Break-Induced Replication (BIR) that involve extensive and mutagenic DNA synthesis spanning even hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here we demonstrate that BIR is suppressed at two-ended DSBs by several proteins coordinating the usage of both DSB ends: ssDNA annealing protein Rad52 and Rad59, D-loop unwinding helicase Mph1, and DSB ends tethering Mre11-Rad50-Xrs2 complex. Finally, BIR is also suppressed when a normally heterochromatic repair template is silenced by Sir2. These findings suggest several mechanisms restricting mutagenic BIR during repair of two-ended DSBs.

genetics↗