Synthesis-dependent Strand Annealing Research Articles

Helicase HELQ: Molecular Characters Fit for DSB Repair Function.

The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA-strand-annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double-strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end-resection-dependent DSB repair pathways, such as homologous recombination (HR), single-strand annealing (SSA), microhomology-mediated end joining (MMEJ), as well as the sub-pathways' synthesis-dependent strand annealing (SDSA) and break-induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Bloom helicase mediates formation of large single\u2013stranded DNA loops during DNA end processing

Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double–strand breaks (DSBs), to yield long 3′ ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single–molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N–terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis–dependent strand annealing (SSA) instead of HR–mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.

Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster.

Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3′ end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in Drosophila melanogaster lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the blm helq and blm fancm double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.

Homology-directed repair involves multiple strand invasion cycles in fission yeast.

Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non–crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent colocalization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in rqh1Δ cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

Helicase Q promotes homology-driven DNA double-strand break repair and prevents tandem duplications

DNA double-strand breaks are a major threat to cellular survival and genetic integrity. In addition to high fidelity repair, three intrinsically mutagenic DNA break repair routes have been described, i.e. single-strand annealing (SSA), polymerase theta-mediated end-joining (TMEJ) and residual ill-defined microhomology-mediated end-joining (MMEJ) activity. Here, we identify C. elegans Helicase Q (HELQ-1) as being essential for MMEJ as well as for SSA. We also find HELQ-1 to be crucial for the synthesis-dependent strand annealing (SDSA) mode of homologous recombination (HR). Loss of HELQ-1 leads to increased genome instability: patchwork insertions arise at deletion junctions due to abortive rounds of polymerase theta activity, and tandem duplications spontaneously accumulate in genomes of helq-1 mutant animals as a result of TMEJ of abrogated HR intermediates. Our work thus implicates HELQ activity for all DSB repair modes guided by complementary base pairs and provides mechanistic insight into mutational signatures common in HR-defective cancers.

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ∼10 nt, but it was required when flaps were ∼500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ∼20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ∼20 nt and is completely required at ∼50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ∼20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

ATRX and RECQ5 define distinct homologous recombination subpathways

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

CRISPR-based strategies for targeted transgene knock-in and gene correction.

The last few years have seen tremendous advances in CRISPR-mediated genome editing. Great efforts have been made to improve the efficiency, specificity, editing window, and targeting scope of CRISPR/Cas9-mediated transgene knock-in and gene correction. In this article, we comprehensively review recent progress in CRISPR-based strategies for targeted transgene knock-in and gene correction in both homology-dependent and homology-independent approaches. We cover homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) pathways for a homology-dependent strategy and alternative DNA repair pathways such as non-homologous end joining (NHEJ), base excision repair (BER), and mismatch repair (MMR) for a homology-independent strategy. We also discuss base editing and prime editing that enable direct conversion of nucleotides in genomic DNA without damaging the DNA or requiring donor DNA. Notably, we illustrate the key mechanisms and design principles for each strategy, providing design guidelines for multiplex, flexible, scarless gene insertion and replacement at high efficiency and specificity. In addition, we highlight next-generation base editors that provide higher editing efficiency, fewer undesired by-products, and broader targeting scope.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Investigating SLX4‐Dependent Recruitment of a DNA Repair Protein to DNA Double Strand Breaks in Dividing Cells

Cancer is a disease that functions as a consequence of abnormal cell growth, generally caused by DNA damage that triggers abnormal cell function. One form of DNA damage is the double‐strand break (DSB) in which both strands of a chromosome are severed in near proximity. In normal cells, several forms of DSB repair, including single strand annealing (SSA) and synthesis dependent strand annealing (SDSA), fix the double strand break (DSB), restoring an intact chromosome, albeit sometimes with an altered DNA sequence. These DNA repair processes require the sequential action of numerous proteins that enable their many steps. In S. cerevisiae these include the Rad1‐Rad10 endonuclease complex, which cleaves nonhomologous DNA flaps in SSA and SDSA as one of the final steps.We have evidence that the Slx4 protein is partially required for the recruitment of Rad1‐Rad10 to DSB sites when cells are dividing (in S, G2 or M phases). Those experiments did not show the basis for the “partial” nature of the recruitment, but our hypothesis is that the Slx4‐dependency is tied to a specific phase of the cell cycle, possibly S phase. A plausible explanation is that when Slx4 exercises its checkpoint signal dampening role in S phase, which promotes homologous recombination, it indirectly recruits Rad1‐Rad10 to double‐strand break repair sites. Since, following DNA damage, checkpoint signal dampening is triggered (in part) by phosphorylation of Slx4 in S phase and the specific phosphorylation sites for this are known, we will test our hypothesis by creating mutant strains which lack these sites and repeating the earlier experiments using synchronized cell cultures. We began creating the mutant strains by cloning DNA plasmids containing the slx4‐7MUT gene, our focus, in which wild‐type SLX4 has been mutated to alanine at the seven phosphorylation sites activating checkpoint signal dampening. Candidate clones were screened by PCR and DNA sequencing. The successful candidate plasmids were transformed into a panel of W303 background yeast strains and integration of the mutant cassettes was confirmed by PCR and DNA sequencing screens. The strains were then crossed to strains bearing the fluorescence microscopy assay markers we will use in the fluorescence microscopy assay being employed. We have carried out preliminary fluorescence microscopy assays in which cultures of the progeny of these crosses were synchronized at the G1/S boundary and released back into the cell cycle that show acceptable arrest but the limited release back into the cell cycle that was observed requires further optimization. Using this separation‐of‐function mutant strain will allow us to compare recruitment of Rad1‐Rad10 in SLX4 wild‐type strains with Slx4 checkpoint signal dampening mutants. Understanding the process of Rad1‐Rad10 recruitment may aid in the design and development of a more effective means of cancer treatment.

Exploring Rad1‐Rad10 Recruitment by Slx4 in Relation to Cell Cycle

DNA damage, caused by various exogenous and endogenous agents such as ionizing radiation and replication stress, gives rise to mutations in DNA that can cause cancer. To combat this, eukaryotic cells use evolutionarily conserved repair mechanisms that mend the DNA. The choice of repair pathway depends primarily on the form of damage, its location within the genome, and the cell cycle phase. For example, single‐strand annealing (SSA), a form of DNA double‐strand break repair (DSBR), is favored in regions between DNA repeats while synthesis‐dependent strand annealing (SDSA), another DSBR mode, needs sister chromatids which act as the template DNA for repair. In order for SSA and SDSA to be completed, the Rad1‐Rad10 endonuclease cleaves overhanging flaps that result from the annealing step before the final ligation can proceed.We have preliminary data indicating that Slx4, a protein present in S. cerevisiae (baker’s yeast), is partially required in the recruitment of Rad1‐Rad10 to repair sites during cell division (S, G2 and M phases). However, cells characterized as being in cell division were not differentiated during data analysis due to the size of the population within those phases. Our research seeks to elucidate the phase in which Slx4 exerts its recruitment properties by repeating our earlier fluorescence microscopy recruitment assay using yeast strains that contain fluorescently‐tagged proteins for monitoring both the colocalization of Rad1‐Rad10 to an induced double‐strand break site and the phase of the cell cycle in cell cycle‐synchronized cultures.To help us clearly distinguish between cell cycle phases during fluorescence microscopy, we have integrated a fluorescently labeled spindle pole body protein (Spc110‐CFP) into our strains because of its dramatic morphology shifts during cell division. Yeast genetic crosses were conducted to combine Spc110‐CFP with SLX4 mutant genes as well as other features that will allow us to perform fluorescence microscopy and flow cytometry analysis in a single strain. Following cell cycle arrest by alpha‐factor, > 90% of cells showed a single Spc110‐CFP focus, indicating that most cells were successfully arrested at the G1/S phase boundary, but flow cytometry analysis showed that only about 60% of cells contained a DNA content consistent with G1/S arrest. These data indicate that cell cycle arrest and its monitoring by the appearance of Spc110‐CFP foci are effective but further efforts are needed to optimize the protocol. Along these lines, we began creating a new SLX4 deletion mutant using Polymerase Chain Reaction (PCR) to amplify a transplacement mutant gene in which the SLX4 coding region has been swapped with the antibiotic marker NATMX4. The final PCR product was A‐tailed and then ligated into the pGEMT‐Easy vector. E. coli transformants were screened by PCR for the insert and will be sequenced to verify the SLX4 region is in the plasmid before moving forward with yeast genetic crosses.Support or Funding InformationNational Institute of Health (NIH) (Grant SC1GM127204), NIH MARC Program (NIH Grant GM008395), California State University, Northridge College of Science and Math, California State University, Northridge Department of Chemistry and Biochemistry

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.

Features of DNA Helicase Encoded by the uvrD Gene of Deinococcus radiodurans R1 in Escherichia coli K-12 Cells

Reparative helicase II of Deinococcus radiodurans performs an unexpected critical function in repair of double-strand DNA breaks through the mechanism of extended synthesis-dependent strand annealing (ESDSA), while it is considered as an optional participant in the RecF pathway of recombinational repair in Escherichia coli. A fragment of genomic DNA of the radioresistant bacterium Deinococcus radiodurans with the uvrD gene encoding DNA helicase II, which is involved in excision repair of nucleotides, mismatch repair, and recombinational repair and replication, was cloned in the cells of the model object, Escherichia coli K-12. The pCR 2.1-uvrD+ plasmid restores resistance to ultraviolet light of mutant cells of Escherichia coliuvrD–, helD–, and rep– defective in reparative helicase II, helicase IV, and replicative helicase Rep, respectively, almost to the level of wild-type AB1157 and uvrD+, and, to a lesser extent, the strain with a mutation in the recQ gene encoding the key helicase of recombinational repair RecQ. The protective effect is also noticeable when strains with the plasmid are irradiated with γ-rays. It is established that Deinococcus radiodurans UvrD helicase possesses broad possibilities.

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote.

Mechanisms that ensure repair of double-strand DNA breaks (DSBs) are instrumental in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found as a concatemer comprising multiple co-integrated transgene copies. Here, we investigated the contribution of various DSB repair pathways to the concatemer formation. We injected mouse zygotes with a pool of linear DNA molecules carrying unique barcodes at both ends and obtained 10 transgenic embryos with 1–300 transgene copies. Sequencing the barcodes allowed us to assign relative positions to the copies in concatemers and detect recombination events that occurred during integration. Cumulative analysis of approximately 1,000 integrated copies reveals that over 80% of them underwent recombination when their linear ends were processed by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR). We also observed evidence of double Holliday junction (dHJ) formation and crossing over during the concatemer formations. Sequencing indels at the junctions between copies shows that at least 10% of DNA molecules introduced into the zygotes are ligated by non-homologous end joining (NHEJ). Our barcoding approach, verified with Pacific Biosciences Single Molecule Real-Time (SMRT) long-range sequencing, documents high activity of homologous recombination after DNA microinjection.

Pathways and assays for DNA double-strand break repair by homologous recombination.

Double strand breaks (DSBs) are the most detrimental type of DNA damage that must be repaired to ensure genome integrity and cell survival. Unrepaired or improperly repaired DSBs can potentially cause tumorigenesis or cell death. DSBs are primarily repaired by non-homologous end joining or homologous recombination (HR). The HR pathway is initiated by processing of the 5'-end of DSBs to generate 3'-end single-strand DNA (ssDNA). Furthermore, the intermediate is channeled to one of the HR sub-pathways, including: (i) double Holliday junction (dHJ) pathway, (ii) synthesis-dependent strand annealing (SDSA), (iii) break-induced replication (BIR), and (iv) single-strand annealing (SSA). In the dHJ sub-pathway, the 3'-ssDNA coated with Rad51 recombinase performs homology search and strand invasion, forming a displacement loop (D-loop). Capture of the second end by the D-loop generates a dHJ intermediate that is subsequently dissolved by DNA helicase or resolved by nucleases, producing non-crossover or crossover products. In SDSA, the newly synthesized strand is displaced from the D-loop and anneals to the end on the other side of the DSBs, producing non-crossovers. In contrast, BIR repairs one-end DSBs by copying the sequence up to the end of the template chromosome, resulting in translocation or loss of heterozygosity. SSA takes place when resection reveals flanking homologous repeats that can anneal, leading to deletion of the intervening sequences. A variety of reporter assays have been developed to monitor distinct HR sub-pathways in both Saccharomyces cerevisiae and mammals. Here, we summarize the principles and representative assays for different HR sub-pathways with an emphasis on the studies in the budding yeast.

Role of the Srs2-Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2–Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2–Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo. Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity.

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.