RecA Homolog Research Articles

SCFRMF mediates degradation of the meiosis-specific recombinase DMC1

Meiotic recombination requires the specific RecA homolog DMC1 recombinase to stabilize strand exchange intermediates in most eukaryotes. Normal DMC1 levels are crucial for its function, yet the regulatory mechanisms of DMC1 stability are unknown in any organism. Here, we show that the degradation of Arabidopsis DMC1 by the 26S proteasome depends on F-box proteins RMF1/2-mediated ubiquitination. Furthermore, RMF1/2 interact with the Skp1 ortholog ASK1 to form the ubiquitin ligase complex SCFRMF1/2. Genetic analyses demonstrate that RMF1/2, ASK1 and DMC1 act in the same pathway downstream of SPO11-1 dependent meiotic DNA double strand break formation and that the proper removal of DMC1 is crucial for meiotic crossover formation. Moreover, six DMC1 lysine residues were identified as important for its ubiquitination but not its interaction with RMF1/2. Our results reveal mechanistic insights into how the stability of a key meiotic recombinase that is broadly conserved in eukaryotes is regulated.

Understanding the structural basis of the binding specificity of c-di-AMP to M. smegmatis RecA using computational biology approach

Mycobacterium tuberculosis RecA (MtRecA), a protein involved in DNA repair, homologous recombination and SOS pathway, contributes to the development of multidrug resistance. ATP binding-site in RecA has been a drug target to disable RecA dependent DNA repair. For the first time, experiments have shown the existence and binding of c-di-AMP to a novel allosteric site in the C-terminal-Domain (CTD) of Mycobacterium smegmatis RecA (MsRecA), a close homolog of MtRecA. In addition, it was observed that the c-di-AMP was not binding to Escherichia coli RecA (EcRecA). This article analyses the possible interactions of the three RecA homologs with the various c-di-AMP conformations to gain insights into the structural basis of the natural preference of c-di-AMP to MsRecA and not to EcRecA, using the structural biology tools. The comparative analysis, based on amino acid composition, homology, motifs, residue types, docking, molecular dynamics simulations and binding free energy calculations, indeed, conclusively indicates strong binding of c-di-AMP to MsRecA. Having very similar results as MsRecA, it is highly plausible for c-di-AMP to strongly bind MtRecA as well. These insights from the in-silico studies adds a new therapeutic approach against TB through design and development of novel allosteric inhibitors for the first time against MtRecA. Communicated by Ramaswamy H. Sarma

The role of conserved amino acid residues of Sae3 in Mei5-Sae3 complex for Dmc1 assembly in meiotic recombination.

Meiotic recombination between homologous chromosomes is promoted by the collaborative action of two RecA homologs, Rad51 and meiosis-specific Dmc1. The filament assembly of Dmc1 is promoted by meiosis-specific Mei5-Sae3 in budding yeast. Mei5-Sae3 shows sequence similarity to fission yeast Sfr1-Swi5, which stimulates DNA strand exchanges by Rad51 as well as Dmc1. Sae3 and Swi5 share a conserved motif with the amino acid sequence YNEI/LK/RD. In this study, we analyzed the role of the YNEL residues in the Sae3 sequence in meiotic recombination and found that these residues are critical for Sae3 function in Dmc1 assembly. L59 substitution in the Sae3 protein disrupts complex formation with Mei5, while Y56 and N57 substitutions do not. These observations reveal the differential contribution of conserved YNEL residues to Sae3 activities in meiotic recombination.

Genome-Wide Identification of the LexA-Mediated DNA Damage Response in Streptomyces venezuelae.

ABSTRACTDNA damage triggers a widely conserved stress response in bacteria called the SOS response, which involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs, but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA-damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA-alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using surface plasmon resonance, we further confirmed the LexA DNA binding motif (SOS box) and demonstrated that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage.IMPORTANCE The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic-producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens.

Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80bp or 1kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.

The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis.

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Reverse breeding: A modern plant breeding approach for hybrid recreation

Reverse breeding is a modern plant breeding method for producing complementing parental lines for any heterozygous plant through achaismatic meiosis (meiosis without crossovers). The achiasmatic meiosis leads to univalent segregation at meiotic metaphase-I and the generation of aneuploid gametes. These gametes are then regenerated as doubled-haploid (DH) plants. Each DH carries combinations of its parental chromosomes, and complementing DH pairs can be crossed to reconstitute the initial hybrid. In reverse breeding, the suppression of meiotic crossovers in a hybrid ensures the transmission of non-recombinant chromosomes to haploid gametes. The PAIR2 gene is required for homologous chromosome synapsis at meiosis-I in plants. An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis thaliana ASY1, results in a defect in homologous chromosome pairing during meiosis, display univalents at metaphase-I. Essentially, reverse breeding follows an approach akin to the generation of a DH population from an F1 hybrid, carrying a dominant-acting transgene that down-regulates the expression of Disrupted Meiotic cDNA1 (DMC1), resulting in inhibition of crossover recombination and thereby enabling intact-chromosome inheritance. In earlier reports on reverse breeding in A. thaliana, a hybrid was constructed of using two of its natural ecotypes (Col-0 and Laer-0), carrying an RNAi transgene targeting the meiotic recombinase (RecA homolog) DMC1 that prevented the formation of meiotic crossover recombination. This method mainly included steps: (i) the generation and selection of RNAi: DMC1 transformed lines; (ii) the generation of achiasmatic hybrids; (iii) the crossing of achiasmatic hybrids to GFP-tailswap to generate haploid chromosome substitution lines (CSLs); (iv) the generation of DHs by spontaneous doubling of haploid CSLs; and (v) the crossing of complementing CSLs to recreate the initial hybrid. The scope of reverse breeding could be envisioned for the improvement of agricultural crops, as it may enable the generation of parental breeding lines for the recreation of hybrid.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis.

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.

Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1.

In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.

A putative human infertility allele of the meiotic recombinase DMC1 does not affect fertility in mice.

Whole-exome or whole-genome sequencing is becoming routine in clinical situations for identifying mutations underlying presumed genetic causes of disease including infertility. While this is a powerful approach for implicating polymorphisms or de novo mutations in genes plausibly related to the phenotype, a greater challenge is to definitively prove causality. This is a crucial requisite for treatment, especially for infertility, in which validation options are limited. In this study, we created a mouse model of a putative infertility allele, DMC1M200V. DMC1 encodes a RecA homolog essential for meiotic recombination and fertility in mice. This allele was originally implicated as being responsible for the sterility of a homozygous African woman, a conclusion supported by subsequent biochemical analyses of the mutant protein and by studies of yeast with the orthologous amino acid change. Here, we found that Dmc1M200V/M200V male and female mice are fully fertile and do not exhibit any gonadal abnormalities. Detailed immunocytological analysis of meiosis revealed no defects suggestive of compromised fertility. This study serves as a cautionary tale for making conclusions about consequences of genetic variants, especially with respect to infertility, and emphasizes the importance of conducting relevant biological assays for making accurate diagnoses in the era of genomic medicine.

Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search.

Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.

Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein

The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51. F86L is a breast tumor-derived somatic variant that affects the interface between adjacent RAD51 protomers in the presynaptic filament. E258A is a germline variant that maps to the interface region between the N-terminal and RecA homology domains of RAD51. Both variants exhibit abnormal biochemistry including altered DNA strand exchange activity. Both variants inhibit the DNA strand exchange activity of wild-type RAD51, suggesting a mechanism for negative dominance. The inhibitory effect of F86L on wild-type RAD51 is surprising since F86L alone exhibits robust DNA strand exchange activity. Our findings indicate that even DNA strand exchange-proficient variants can have negative functional interactions with wild-type RAD51. Thus heterozygous F86L or E258 mutations in RAD51 could promote genomic instability, and thereby contribute to tumor progression.

The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

RecN is a cohesin-like protein involved in DNA double-strand break repair in bacteria. The RecA recombinase functions to mediate repair via homologous DNA strand invasion to form D-loops. Here we provide evidence that the RecN protein stimulates the DNA strand invasion step of RecA-mediated recombinational DNA repair. The intermolecular DNA tethering activity of RecN protein described previously cannot fully explain this novel activity since stimulation of RecA function is species-specific and requires RecN ATP hydrolysis. Further, DNA-bound RecA protein increases the rate of ATP hydrolysis catalysed by RecN during the DNA pairing reaction. DNA-dependent RecN ATPase kinetics are affected by RecA protein in a manner suggesting a specific order of protein–DNA assembly, with RecN acting after RecA binds DNA. We present a model for RecN function that includes presynaptic stimulation of the bacterial repair pathway perhaps by contributing to the RecA homology search before ternary complex formation.

Exploring the Role of ATP Hydrolysis in the RecA Protein via Stalled DNA Intermediates

A central participant in the recombinational DNA‐repair system is the recombinase protein, RecA, which acts as a DNA pairing enzyme that is responsible for promoting an exchange of homologous DNA strands. Physical studies have revealed three DNA binding sites buried within the interior of the RecA filament. This current understanding of RecA is incomplete as it fails to explain how a four‐strand DNA exchange occurs between two duplex DNA fragments.The bacterial RecA homologue is unique in that it couples ATP‐hydrolysis with DNA strand exchange. ATP hydrolysis occurrs uniformly throughout the RecA filament, with only a small fraction of ATP used for filament disassembly. The mechanism by which the RecA filament utilizes the rest of ATP hydrolysis to perform DNA strand exchange reactions is not fully understood. My aim is to explore the hypothesis that the majority of ATP utilized by the reaction is hydrolyzed at binding sites located longitudinally along both the interior and exterior of the filament, providing the energy needed to rotate one segment of DNA around another.This will be explored by performing RecA mediated strand‐exchange reactions with a variety of linear, double stranded DNA substrates containing heterologous regions. When the RecA protein is run into a heterologous barrier, a stalled DNA‐protein intermediate molecule is the product. Formation of these intermediates has been obtained. Imaging with Electron Microscopy shows topological changes that offer fundamental insight into the universal mechanics of RecA‐mediated strand exchanges. Furthermore, evidence shows that introduction of the protein Uup modifies the structure of the intermediates, providing insight into Uup's function.

Ganetespib overcomes resistance to PARP inhibitors in breast cancer by targeting core proteins in the DNA repair machinery.

DNA damage repair plays essential roles in drug resistance, especially resistance to Poly (ADP-ribose) polymerase (PARP) inhibitors in the clinic. A subset of DNA repair proteins such as Breast cancer gene 1 (BRCA1), BRCA2 and RecA homolog (RAD51) are client proteins of heat shock protein 90 (Hsp90). Clearance of these DNA repair proteins by inhibition of Hsp90 is a promising strategy for overcoming resistance to PARP inhibitors. Here we report the pharmacological analysis of the highly potent second-generation Hsp90 inhibitor, ganetespib. Methods Nuclear BRCA1, BRCA2, and RAD51 expression in breast cancer cells were detected by subcellular fractionation and western blot analysis. Formation of nuclear RAD51 and γ-H2AX foci was analyzed by immunofluorescent staining. The cytotoxicity of ganetespib and ABT-888 in breast cancer cells were evaluated by cell proliferation, colony survival, and apoptosis assay. To investigate the efficacy of this therapy in vivo, SCID mice bearing MCF7 xenografts were treated with ganetespib and ABT-888, both as single agents and in combination. Results Ganetespib significantly destabilized nuclear BRCA1, BRCA2, and RAD51, and efficiently disrupted homologous recombination-mediated DNA double-strand break repair in breast cancer cells. The synergistic antitumor effects of ganetespib and the PARP inhibitor, ABT-888 were observed, and concurrent treatment with both inhibitors synergistically inhibited xenograft tumor growth. Importantly, the combined treatment was well tolerated, without significant loss of body weight or major histological changes in the breast cancer xenograft model. Conclusion These data provide a novel strategy for the treatment of breast cancer with wild type BRCA1 using combination therapy targeting Hsp90 to overcome resistance to PARP inhibitors.

Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts.

The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss Physcomitrella patens Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity. Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner. Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement. This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the mechanisms underlying these processes may be intimately related in C. reinhardtii cps.

Evolution of bacterial recombinase A (recA) in eukaryotes explained by addition of genomic data of key microbial lineages.

Recombinase enzymes promote DNA repair by homologous recombination. The genes that encode them are ancestral to life, occurring in all known dominions: viruses, Eubacteria, Archaea and Eukaryota. Bacterial recombinases are also present in viruses and eukaryotic groups (supergroups), presumably via ancestral events of lateral gene transfer. The eukaryotic recA genes have two distinct origins (mitochondrial and plastidial), whose acquisition by eukaryotes was possible via primary (bacteria-eukaryote) and/or secondary (eukaryote-eukaryote) endosymbiotic gene transfers (EGTs). Here we present a comprehensive phylogenetic analysis of the recA genealogy, with substantially increased taxonomic sampling in the bacteria, viruses, eukaryotes and a special focus on the key eukaryotic supergroup Amoebozoa, earlier represented only by Dictyostelium We demonstrate that several major eukaryotic lineages have lost the bacterial recombinases (including Opisthokonta and Excavata), whereas others have retained them (Amoebozoa, Archaeplastida and the SAR-supergroups). When absent, the bacterial recA homologues may have been lost entirely (secondary loss of canonical mitochondria) or replaced by other eukaryotic recombinases. RecA proteins have a transit peptide for organellar import, where they act. The reconstruction of the RecA phylogeny with its EGT events presented here retells the intertwined evolutionary history of eukaryotes and bacteria, while further illuminating the events of endosymbiosis in eukaryotes by expanding the collection of widespread genes that provide insight to this deep history.

Hop2 and Sae3 Are Required for Dmc1-Mediated Double-Strand Break Repair via Homolog Bias during Meiosis.

During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the hop2Δ or sae3Δ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.