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1.  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.
doi:10.1093/nar/gkt138
PMCID: PMC3632125  PMID: 23460207
2.  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.
doi:10.1091/mbc.E07-05-0490
PMCID: PMC1995734  PMID: 17671161
3.  RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation 
The Journal of Cell Biology  2009;185(4):587-600.
The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.
doi:10.1083/jcb.200811079
PMCID: PMC2711581  PMID: 19451272
4.  Interaction of TATA-Binding Protein with Upstream Activation Factor Is Required for Activated Transcription of Ribosomal DNA by RNA Polymerase I in Saccharomyces cerevisiae In Vivo 
Molecular and Cellular Biology  1998;18(7):3752-3761.
Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations in RRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations, L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.
PMCID: PMC108958  PMID: 9632758
5.  Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30▿ †  
Molecular and Cellular Biology  2008;28(21):6709-6719.
Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ∼70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.
doi:10.1128/MCB.00703-08
PMCID: PMC2573240  PMID: 18765638
6.  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.
doi:10.1111/j.1365-2958.2011.07703.x
PMCID: PMC3170485  PMID: 21615552
7.  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.
doi:10.1091/mbc.E10-01-0050
PMCID: PMC2893993  PMID: 20444977
8.  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
9.  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.
doi:10.1128/MCB.00870-13
PMCID: PMC3838189  PMID: 24001775
10.  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.
doi:10.1371/journal.pgen.1003971
PMCID: PMC3836719  PMID: 24278037
11.  Rad22Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease 
Molecular Biology of the Cell  2006;17(4):2081-2090.
Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate ∼50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number.
doi:10.1091/mbc.E05-11-1006
PMCID: PMC1415312  PMID: 16467377
12.  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.
doi:10.1371/journal.pone.0081371
PMCID: PMC3855272  PMID: 24339919
13.  Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP 
Molecular and Cellular Biology  2001;21(7):2292-2297.
Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9 switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269–5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5, RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF component RRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.
doi:10.1128/MCB.21.7.2292-2297.2001
PMCID: PMC86863  PMID: 11259579
14.  Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair 
The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
doi:10.1128/MMBR.66.4.630-670.2002
PMCID: PMC134659  PMID: 12456786
15.  Peptide aptamer mimicking RAD51-binding domain of BRCA2 inhibits DNA damage repair and survival in Trypanosoma brucei 
Microbial pathogenesis  2011;50(5):252-262.
The eukaryotic DNA recombination repair protein BRCA2 is functional in the parasitic protozoan Trypanosoma brucei. The mechanism of the involvement of BRCA2 in homologous recombination includes its interaction with the DNA recombinase proteins of the RAD51 family. BRCA2 is known to interact with RAD51 through its unique and essential BRC sequence motifs. T. brucei BRCA2 homolog (TbBRCA2) has fifteen repeating BRC motifs as compared to mammalian BRCA2 that has only eight. We report here our yeast 2-hybrid analysis studies on the interactions of TbBRCA2 BRC motifs with five different RAD51 paralogues of T. brucei. Our study revealed that a single BRC motif is sufficient to bind to these RAD51 paralogues. To test the possibility whether a single 44 amino acid long repeating unit of the TbBRCA2 BRC motif may be exploited as an inhibitor of T. brucei growth, we ectopically expressed this peptide segment in the procyclic form of the parasite and evaluated its effects on cell survival as well as the sensitivity of these cells to the DNA damaging agent methyl methane sulfonate (MMS). Expression of a single BRC motif led to MMS sensitivity and inhibited cellular proliferation in T. brucei.
doi:10.1016/j.micpath.2010.11.007
PMCID: PMC3065951  PMID: 21296653
Trypanosoma brucei; BRCA2; RAD51; homologous recombination; DNA repair; Methyl methane sulfonate; yeast 2-hybrid analysis; aptamer
16.  Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2199-9-35
PMCID: PMC2324109  PMID: 18402694
17.  Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA 
Molecular cell  2009;35(1):105-115.
SUMMARY
Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR.
doi:10.1016/j.molcel.2009.05.026
PMCID: PMC2711036  PMID: 19595720
18.  Characterization of the fission yeast ribosomal DNA binding factor: components share homology with Upstream Activating Factor and with SWI/SNF subunits 
Nucleic Acids Research  2002;30(24):5347-5359.
A ribosomal DNA (rDNA) binding activity was previously characterized in fission yeast that recognized the upstream ribosomal RNA (rRNA) gene promoter in a sequence specific manner and which stimulated rRNA synthesis. It was found to share characteristics with Saccharomyces cerevisiae’s Upstream Activating Factor (UAF), an RNA polymerase I (pol I) specific transcription stimulatory factor. Putative fission yeast homologs of the S.cerevisiae UAF subunits, Rrn5p and Rrn10p, were identified. The Schizosaccharomyces pombe rDNA binding activity/transcriptional stimulatory activity was found to co-fractionate with both SpRrn5h and SpRrn10h. Analysis of polypeptides interacting with SpRrn10h uncovered a 27 kDa polypeptide (Spp27) homologous to a SWI/SNF component (now known to be homologous to Uaf30p). The contributions of the S.pombe and S.cerevisiae upstream rDNA promoter domains were assessed in cross-species transcriptional assays. Furthermore, comparative genomic analysis revealed putative Rrn5p, Rrn10p, Rrn9p and p27 homologs in multiple non-vertebrates. The S.pombe rDNA binding activity is proposed to be an RNA pol I specific SWI/SNF type factor.
PMCID: PMC140063  PMID: 12490702
19.  RAD51 localization and activation following DNA damage. 
The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.
PMCID: PMC1693300  PMID: 15065660
20.  Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities 
Nucleic Acids Research  2009;37(13):4296-4307.
RAD51, a eukaryotic recombinase, catalyzes homologous-pairing and strand-exchange reactions, which are essential steps in homologous recombination and recombinational repair of double strand breaks. On the other hand, human PSF was originally identified as a component of spliceosomes, and its multiple functions in RNA processing, transcription and DNA recombination were subsequently revealed. In the present study, we found that PSF directly interacted with RAD51. PSF significantly enhanced RAD51-mediated homologous pairing and strand exchange at low RAD51 concentrations; however, in contrast, it inhibited these RAD51-mediated recombination reactions at the optimal RAD51 concentration. Deletion analyses revealed that the N-terminal region of PSF possessed the RAD51- and DNA-binding activities, but the central region containing the RNA-recognition motifs bound neither RAD51 nor DNA. These results suggest that PSF may have dual functions in homologous recombination and RNA processing through its N-terminal and central regions, respectively.
doi:10.1093/nar/gkp298
PMCID: PMC2715225  PMID: 19447914
21.  Correlation between suppressed meiotic recombination and the lack of DNA strand-breaks in the rRNA genes of Saccharomyces cerevisiae. 
Nucleic Acids Research  1984;12(18):7199-7213.
We have examined whether the suppressed homologous meiotic recombination within the rDNA of S. cerevisiae is reflected by a lack of possibly recombination-initiating strand-breaks in this part of the genome. Our findings indicate that bulk DNA in the ds-break repair deficient mutant rad52/rad52 accumulates a limited number of both ss- and ds-breaks during meiosis as compared to a RAD+/rad52 heterozygote. The rDNA-containing chromosome is however protected against these breaks, and thus this may be an explanation for the suppression of recombination in the rDNA. The fact that ds-breaks seem to be involved gives indirect support to the ds-break-repair model for recombination.
PMCID: PMC320151  PMID: 6384933
22.  Interrogation of the Protein-Protein Interactions between Human BRCA2 BRC Repeats and RAD51 Reveals Atomistic Determinants of Affinity 
PLoS Computational Biology  2011;7(7):e1002096.
The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.
Author Summary
The atomic scale interactions that occur at the interfaces between proteins are fundamental to all biological processes. One such critical interface is formed between the proteins, human BRCA2 and RAD51. BRCA2 binds to and delivers RAD51 to sites of DNA damage, where RAD51 mediates the error-free repair of double-stranded DNA breaks. Mutations in BRCA2 have been linked to breast cancer predisposition. Therefore, an accurate picture of the interactions between these two proteins is of great importance. BRCA2 interacts with RAD51 via eight “BRC repeats” that are similar, but not identical, in sequence. Due to lack of experimental structural information regarding the binding of seven of the eight BRC repeats to RAD51, it is unknown how subtle sequence variations in the repeats translate to measurable variations in their binding affinity. We have used a range of computational methods, firstly based on classical force fields, and secondly based on first principles quantum mechanical techniques whose computational cost scales linearly with the number of atoms, allowing us to perform calculations on the entire protein complex. This is the first study comparing all eight BRC repeats at the atomic scale and our results provide critical insights into the control of RAD51 by human BRCA2.
doi:10.1371/journal.pcbi.1002096
PMCID: PMC3136434  PMID: 21789034
23.  Physical Interaction of RECQ5 Helicase with RAD51 Facilitates Its Anti-recombinase Activity* 
The Journal of Biological Chemistry  2010;285(21):15739-15745.
Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.
doi:10.1074/jbc.M110.110478
PMCID: PMC2871440  PMID: 20348101
Chromosomes; DNA Damage; DNA Helicase; DNA Recombination; DNA Repair; DNA Double-Strand Break; Genomic Instability; RAD51; RecQ
24.  Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein 
Nucleic Acids Research  2006;34(16):4429-4437.
The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.
doi:10.1093/nar/gkl562
PMCID: PMC1636354  PMID: 16945962
25.  Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo 
The Journal of Cell Biology  2009;185(6):969-981.
Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 “anti-recombinase” restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.
doi:10.1083/jcb.200810055
PMCID: PMC2711611  PMID: 19506039

Results 1-25 (978750)