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1.  The Shu complex regulates Rad52 localization during rDNA repair Running Head: Shu1 promotes rDNA repair 
DNA repair  2013;12(9):10.1016/j.dnarep.2013.05.003.
The Shu complex, consisting of Rad51 paralogues, is an important regulator of homologous recombination, an error-free DNA repair pathway. Consequently, when members of this complex are disrupted, cells exhibit a mutator phenotype, sensitivity to DNA damage reagents and increased gross chromosomal rearrangements. Previously, we found that the Shu complex plays an important role in ribosomal DNA (rDNA) recombination when the Upstream Activating Factor (UAF) protein Uaf30 is disrupted. UAF30 encodes a protein needed for rDNA transcription and when deleted, rDNA recombination increases and the rDNA expands in a Shu1-dependent manner. Here we find using the uaf30-sensitized background that the central DNA repair protein Rad52, which is normally excluded from the nucleolus, frequently overlaps with the rDNA. This close association of Rad52 with the rDNA is dependent upon Shu1 in a uaf30 mutant. Previously, it was shown that in the absence of Rad52 sumoylation, Rad52 foci mislocalize to the nucleolus. Interestingly, here we find that using the uaf30 sensitized background the ability to regulate Rad52 sumoylation is important for Shu1 dependent rDNA recombination as well as Rad52 close association with rDNA. Our results suggest that in the absence of UAF30, the Shu complex plays a central role in Rad52 rDNA localization as long as Rad52 can be sumoylated. This discrimination is important for rDNA copy number homeostasis.
PMCID: PMC3811918  PMID: 23790361
DNA repair; UAF complex; Shu complex; Rad52; Rad52 sumoylation; rDNA recombination
2.  The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55–Rad57 to mediate error-free recombination 
Nucleic Acids Research  2013;41(8):4525-4534.
The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2–Psy3 heterodimer preferentially binds synthetic forked DNA or 3′-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55–Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55–Rad57. Importantly, Csm2’s interaction with Rad51 is dependent on Rad55, whereas Csm2’s interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2–Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55–Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.
PMCID: PMC3632125  PMID: 23460207
3.  The Yeast Shu Complex Utilizes Homologous Recombination Machinery for Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue 
PLoS ONE  2013;8(12):e81371.
DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.
PMCID: PMC3855272  PMID: 24339919
4.  Shu Proteins Promote the Formation of Homologous Recombination Intermediates That Are Processed by Sgs1-Rmi1-Top3 
Molecular Biology of the Cell  2007;18(10):4062-4073.
CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination repair (HRR), their precise role(s) within this pathway remains poorly understood. Here, we have identified a specific role for the Shu proteins in a Rad51/Rad54-dependent HRR pathway(s) to repair MMS-induced lesions during S-phase. We show that, although mutation of RAD51 or RAD54 prevented the formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex.
PMCID: PMC1995734  PMID: 17671161
5.  Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2 
Mutation research  2011;714(1-2):33-43.
The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1Δ mus81Δ synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1Δ rnh202Δ synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.
PMCID: PMC3162113  PMID: 21741981
Recombinational repair; SGS1; MUS81; RNase H2
6.  Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae 
Molecules and Cells  2013;36(5):446-454.
Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias.
PMCID: PMC3887942  PMID: 24213600
homolog bias; meiotic recombination; Mek1 kinase; Shu1; Srs2
7.  Shigella dysenteriae ShuS Promotes Utilization of Heme as an Iron Source and Protects against Heme Toxicity 
Journal of Bacteriology  2005;187(16):5658-5664.
Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.
PMCID: PMC1196095  PMID: 16077111
8.  The Smc5/6 Complex and Esc2 Influence Multiple Replication-associated Recombination Processes in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2010;21(13):2306-2314.
This work shows that Mph1, Mms2, and the Shu complex function in distinct pathways in replication-associated recombinational repair and that the Smc5/6 complex and Esc2 prevent the accumulation of toxic recombination intermediates generated in these pathways.
Replication-associated recombinational repair is important for genome duplication and cell survival under DNA damage conditions. Several nonclassical recombination factors have been implicated in this process, but their functional relationships are not clear. Here, we show that three of these factors, Mph1, Mms2, and the Shu complex, can act independently to promote the formation of recombination intermediates during impaired replication. However, their functions become detrimental when cells lack the Smc5/6 complex or Esc2. We show that mph1Δ, mms2Δ, and shu1Δ suppress the sensitivity to the replication-blocking agent methylmethane sulfonate (MMS) in smc6 mutants, with double deletions conferring stronger suppression. These deletion mutations also rescue the MMS sensitivity of esc2Δ cells. In addition, two-dimensional gel analysis demonstrates that mph1Δ, mms2Δ, and shu1Δ each reduce the level of recombination intermediates in an smc6 mutant when cells replicate in the presence of MMS, and that double deletions lead to a greater reduction. Our work thus suggests that Mph1, Mms2, and the Shu complex can function in distinct pathways in replication-associated recombinational repair and that the Smc5/6 complex and Esc2 prevent the accumulation of toxic recombination intermediates generated in these processes.
PMCID: PMC2893993  PMID: 20444977
9.  Rad51 paralogs Rad55-Rad57 balance the anti-recombinase Srs2 in Rad51 filament formation 
Nature  2011;479(7372):245-248.
Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements, and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyzes the signature reactions of recombination, homology search and DNA strand invasion 1,2. Eukaryotes also possess Rad51 paralogs, whose exact role in recombination remains to be defined 3. Here we show that the budding yeast Rad51 paralogs, the Rad55-Rad57 heterodimer, counteract the anti-recombination activity of the Srs2 helicase. Rad55-Rad57 associate with the Rad51-ssDNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51/Rad55-Rad57 co-filament resists disruption by the Srs2 anti-recombinase by blocking Srs2 translocation involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogs in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 anti-recombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogs, which are known to be involved in cancer predisposition and human disease.
PMCID: PMC3213327  PMID: 22020281
10.  Identification of shuA, the gene encoding the heme receptor of Shigella dysenteriae, and analysis of invasion and intracellular multiplication of a shuA mutant. 
Infection and Immunity  1997;65(12):5358-5363.
shuA encodes a 70-kDa outer membrane heme receptor in Shigella dysenteriae. Analysis of the shuA DNA sequence indicates that this gene encodes a protein with homology to TonB-dependent receptors of gram-negative bacteria. Transport of heme by the ShuA protein requires TonB and its accessory proteins ExbB and ExbD. The shuA DNA sequence contains a putative Fur box overlapping the -10 region of a potential shuA promoter, and the expression of shuA is repressed by exogenous iron or hemin in a Fur-dependent manner, although hemin repressed expression to a lesser extent than iron salts. Disruption of this open reading frame on the S. dysenteriae chromosome by marker exchange yielded a strain that failed to use heme as an iron source, indicating that shuA is essential for heme transport in S. dysenteriae. However, shuA is not essential for invasion or multiplication within cultured Henle cells; the shuA mutant invaded and produced normal plaques in confluent cell monolayers.
PMCID: PMC175774  PMID: 9393841
11.  Reconstitution of DNA Strand Exchange Mediated by Rhp51 Recombinase and Two Mediators 
PLoS Biology  2008;6(4):e88.
In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA
Author Summary
Homologous recombination promotes genetic diversity in the next generation and serves as a driving force for evolution. It also provides efficient machinery for repairing DNA damage such as double-strand breaks. Homologous recombination involves DNA exchange between homologous chromosomes, which is mediated by evolutionarily conserved proteins called recombinases. It is thought that a recombinase binds to single-stranded DNA (ssDNA) to form a nucleoprotein filament called the presynaptic filament, and that this higher order structure engages in a search for homologous DNA sequences. Once a homologous duplex is found, the presynaptic filament initiates strand exchange. However, when ssDNA regions are created, they are immediately covered by replication protein A (RPA), thereby inhibiting recombinase filament formation even under conditions in which homologous recombination is appropriate. Previous studies suggested that mediator proteins help load recombinases onto ssDNA, and further studies showed that at least two mediators function together in a single recombination pathway. How these mediators coordinate recombinase loading has been unclear. We have addressed this question by reconstituting an in vitro strand exchange reaction with purified proteins including a fission yeast recombinase, Rhp51, two mediators, Rad22 and the Swi5-Sfr1 complex, and RPA. Our results indicate that Rad22 orchestrates the loading of Rhp51 onto RPA-coated ssDNA by acting as a scaffold for nucleating the recombinase filament, whereas the other mediator, Swi5-Sfr1, stabilizes and activates the filament.
A test tube reconstitution of the strand exchange reaction provides further evidence that the two mediators cooperatively promote the nucleation of active recombinase-ssDNA filaments, creating a higher order structure for recombination.
PMCID: PMC2292753  PMID: 18416603
12.  Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1 
PLoS Genetics  2014;10(8):e1004542.
Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.
Author Summary
Homologous recombination is required for repairing DNA double-strand breaks (DSBs), which are induced by exogenous factors such as DNA damaging agents or by endogenous factors such as collapse of DNA replication fork in mitotic cells. If improperly processed, DSBs could lead to chromosome rearrangement, cell death, or tumorigenesis in mammals, and thus HR is strictly controlled at several steps, including Rad51 recombinase-driven DNA strand exchange reaction. Specifically, DNA helicases have been shown to be important for suppression of inappropriate recombination events. In this study, we analyzed one such DNA helicase, fission yeast Fbh1. We used an in vivo single-DSB repair assay to show that Fbh1 suppresses crossover formation between homologous chromosomes. Next, we obtained in vitro evidence that Fbh1 acts as an inhibitor of the strand-exchange reaction in the absence of Swi5-Sfr1, but stimulates the reaction after it starts. Furthermore, we found that SCFFbh1 has ubiquitin-ligase activity toward Rad51 in vitro and that Fbh1 regulates the protein level of Rad51 in the stationary phase. These results suggest Fbh1 regulates Rad51-mediated homologous recombination by its seemingly-unrelated two activities, DNA helicase/translocase and ubiquitin ligase.
PMCID: PMC4148199  PMID: 25165823
13.  Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination 
PLoS Genetics  2013;9(11):e1003971.
The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.
Author Summary
The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. Through modulation of the activity of the recombinase RAD51, the RAD51 paralogue proteins play key roles in the regulation of recombination. Considerable advances have been made in understanding of the RAD51 paralogue proteins and their roles in mediating RAD51-mediated homologous recombination, however very little is known of possible roles that they may have in other recombination pathways. Working with the plant, Arabidopsis thaliana, we show here major roles for three RAD51 paralogues in RAD51-independent single-strand annealing recombination. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.
PMCID: PMC3836719  PMID: 24278037
14.  Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2 
PLoS Genetics  2013;9(10):e1003833.
The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.
Author Summary
Homologous recombination (HR) is essential for double-strand break repair and participates in post-replication restart of stalled and collapsed replication forks. However, HR can lead to genome rearrangements and has to be strictly controlled. The budding yeast Srs2 is involved in the prevention of high crossing-over frequencies and in the inhibition of HR at replication forks. Nevertheless, important phenotypes of srs2Δ mutants, like sensitivity to DNA damage and synthetic lethality with replication and recombination mutants, can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. The nature of these intermediates remains to be defined. In a screen designed to uncover mutations able to suppress srs2Δ phenotypes, we isolated a novel allele of Rad52 (rad52-L264P), the gene that codes for the major Rad51 nucleoprotein filament mediator. Interestingly, we observed that rad52-L264P bypasses the requirement for Srs2 without affecting DNA repair by HR. We also found that Rad52-L264P specifically prevents the formation of unproductive Rad51 filaments before strand invasion, allowing us to define Srs2 substrates. Further analysis showed that Rad52-L264P mimics the properties of the Rad52-SUMO conjugate, revealing that Rad52 assembles Rad51 filaments differently according to its sumoylation status.
PMCID: PMC3794917  PMID: 24130504
15.  Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica 
In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. RAD52 epistasis group genes involved in recombinational DNA repair, including mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 and rad59 genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in Entamoeba histolytica the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.
In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote E. histolytica using UV-C (150 J/m2) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In E. histolytica genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the E. histolytica RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear foci-like structures in E. histolytica trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands in vitro.
E. histolytica genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear foci-like structures. Functional assays confirmed that EhRAD51 is a bonafide recombinase. These data provided the first insights about the potential roles of the E. histolytica RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.
PMCID: PMC2324109  PMID: 18402694
16.  Functional Characterization of the Shigella dysenteriae Heme ABC Transporter† 
Biochemistry  2008;47(31):10.1021/bi801005u.
The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.
PMCID: PMC3832205  PMID: 18616281
17.  The USP1/UAF1 Complex Promotes Double-Strand Break Repair through Homologous Recombination ▿ † 
Molecular and Cellular Biology  2011;31(12):2462-2469.
Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1−/−, UAF1−/−/−, and USP1−/− UAF1−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.
PMCID: PMC3133424  PMID: 21482670
18.  Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks 
PLoS Genetics  2014;10(1):e1004005.
Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.
Author Summary
Sexual reproduction involves the generation of chromosomally balanced gametes through the specialized cell division of meiosis. A critical component of meiosis is the physical connection of homologous chromosomes through a combination of recombination and sister chromatid cohesion that is necessary for proper chromosome segregation at the first meiotic division. Meiotic recombination is initiated by the introduction of programmed double strand breaks (DSBs) that are processed and bound by RecA-like proteins called recombinases. In vegetative cells, the Rad51 recombinase preferentially mediates strand invasion of sister chromatids, while in meiotic cells, the meiosis-specific Dmc1 recombinase preferentially invades homologs. How Rad51 and Dmc1 activities are coordinated to generate interhomolog recombinants is a key question in meiosis. This work demonstrates that down-regulation of Rad51 activity is important when interhomolog recombination is occurring to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs. Premature activation of Rad51 results in increased intersister recombination and chromosome missegregation, producing inviable gametes. The evolutionary conservation of both Rad51 and Dmc1 suggests that down-regulation of Rad51 during meiosis may be important in metazoans as well as yeast.
PMCID: PMC3900393  PMID: 24465215
19.  Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage 
Molecular Cancer  2015;14(1):33.
In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage.
We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage.
A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420–520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420–520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency.
USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420–520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0311-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4326527
20.  Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7. 
Journal of Bacteriology  1995;177(11):3004-3009.
Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.
PMCID: PMC176986  PMID: 7768795
21.  Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation 
Molecular Microbiology  2011;81(2):434-456.
Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.
PMCID: PMC3170485  PMID: 21615552
22.  Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break 
PLoS Genetics  2015;11(1):e1004928.
The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.
Author Summary
DNA double strand breaks (DSBs) are among the most deleterious types of damage occurring in the genome, as failure to repair these lesions through either non-homologous-end-joining (NHEJ) or homologous recombination (HR) leads to genetic instability. The 5′ strand of a DSB can be nucleolytically degraded by several nucleases and associated factors, including Mre11, CtIP/Sae2, Exo1 and Dna2 together with Bloom helicase/Sgs1, through a finely regulated process called DSB resection. Once resection is initiated, error-prone NHEJ is prevented. Several findings suggest that DSB resection is a double-edged sword, if not finely regulated, since on one hand it is needed for faithful HR, but on the other it may lead to extensive DNA deletions associated with genome instability. Both in mammals and yeast, 53BP1/Rad9 protein binds near the lesion and counteracts the resection process, limiting the formation of ssDNA. By using S. cerevisiae as a model organism, here we show that Rad9 oligomers block the removal of hypo-active Mre11 protein from a persistent DSB, thus limiting initiation of resection and the recruitment of the recombination factor Rad52, in the absence of Sae2. Altogether, these findings pinpoint a critical role of 53BP1/Rad9 in balancing HR and NHEJ repair events throughout the cell cycle.
PMCID: PMC4287487  PMID: 25569305
23.  Inactivation of Uaf1 Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice 
Molecular and Cellular Biology  2013;33(22):4360-4370.
The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1−/− mice. Unexpectedly, homozygous Uaf1−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.
PMCID: PMC3838189  PMID: 24001775
24.  SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks 
PLoS Biology  2009;7(12):e1000252.
SUMO modification of BLM controls the switch between BLM's pro- and anti-recombinogenic roles in homologous recombination following DNA damage during replication.
The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased γ-H2AX foci. Because the increased γ-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess γ-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.
Author Summary
Replication is the process in which cellular DNA is duplicated. DNA damage incurred during replication is detrimental to the cell. Homologous recombination, in which DNA sequences are exchanged between two similar or identical strands of DNA, plays a pivotal role in correcting replication processes that have failed due to DNA breakage and is tightly regulated, because deficient or excess recombination results in genomic instability. Previous studies have implicated the DNA-processing enzyme BLM in the regulation of homologous recombination; BLM is defective in Bloom's syndrome, which is characterized by excess recombination and cancer susceptibility. Here, we show that modification of BLM by the small protein SUMO controls BLM's function in regulating homologous recombination at sites where DNA replication failed. We showed that cells expressing a SUMO-deficient mutant of BLM accumulated more DNA damage and displayed defects in repair by homologous recombination. An enzyme involved in homologous recombination, RAD51, displayed a defect in localization to sites where DNA replication failed. Our data support a model in which SUMO modification regulates BLM's function in homologous recombination by controlling the localization of RAD51 to failed replication sites.
PMCID: PMC2779653  PMID: 19956565
25.  The Recombinases Rad51 and Dmc1 Play Distinct Roles in DNA Break Repair and Recombination Partner Choice in the Meiosis of Tetrahymena 
PLoS Genetics  2011;7(3):e1001359.
Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3′ single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA–strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB–dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.
Author Summary
Sexual reproduction relies on meiosis, the specialized cell division that allows diploid organisms to halve their chromosome content, resulting in the production of gametes containing one copy of each chromosome. In humans, defects in meiosis cause infertility, stillbirths, and congenital diseases. Homologous recombination is a key step in the meiotic program and is essential for maintaining the fidelity of segregation and for creating genetic diversity. Meiotic recombination begins with self-inflicted DNA breaks that are repaired using the homologous chromosome as a template, in a process that depends upon the universal repair protein Rad51 and its meiosis-specific homologue, Dmc1. The relative contributions of Rad51 and Dmc1 to homologous recombination differ among yeasts, plants, and mammals. We have undertaken a study of these proteins in the evolutionarily distant model organism Tetrahymena thermophila with the hope of clarifying the specialization of these recombinases throughout eukaryotic evolution. We show that, while Rad51 is required for DNA repair, only Dmc1 localizes prominently to meiotic DNA break sites. Also, repair via the homologous chromosome depends on Dmc1. These results suggest that nucleoprotein filaments consisting of primarily Dmc1p allow efficient interhomologue repair, while shorter Rad51 filaments may suffice for repair via the sister chromatid.
PMCID: PMC3069121  PMID: 21483758

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