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1.
《DNA Repair》2016
The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex. 相似文献
2.
REV1是跨损伤聚合酶Y家族的重要成员之一,它不仅作为支架蛋白介导Y家族聚合酶招募至损伤位点完成跨损伤DNA合成(translesion DNA synthesis, TLS),还可利用自身的dCMP转移酶活性在一些损伤位点对侧整合dCMP参与TLS。此外,REV1也被报导参与调控同源重组修复。为进一步探讨REV1互作蛋白RAD51和RAD51C在其参与的同源重组修复通路中的调控作用,本研究采用脉冲氮激光微辐射实验,发现RAD51可调控REV1到双链断裂位点的募集。同时,免疫荧光实验结果证明REV1也反过来影响RAD51应答CPT损伤。然而敲低RAD51C并不影响REV1到DNA双链断裂位点的招募。结果表明,REV1和RAD51在HR通路中存在彼此相互调控的关系。 相似文献
3.
Dunlop MH Dray E Zhao W Tsai MS Wiese C Schild D Sung P 《The Journal of biological chemistry》2011,286(43):37328-37334
Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1. 相似文献
4.
Elena Pires Neelam Sharma Platon Selemenakis Bo Wu Yuxin Huang Dauren S. Alimbetov Weixing Zhao Claudia Wiese 《The Journal of biological chemistry》2021,297(1)
RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament. 相似文献
5.
Scott Cukras Euiho Lee Emily Palumbo Pamela Benavidez George-Lucian Moldovan 《Cell cycle (Georgetown, Tex.)》2016,15(19):2636-2646
USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1. 相似文献
6.
RAD51 and RAD54, members of the RAD52 epistasis group, play key roles in homologous recombination (HR). The efficiency of homologous recombination (HR) can be increased by over-expression of either of them. A vector that allows co-expression of RAD51 and RAD54 was constructed to investigate interactions between the two proteins during extra-chromosomal HR. The efficiency of extra-chromosomal HR evaluated by GFP extra-chromosomal HR was enhanced (110-245%) in different transfected Human sarcoma (HT-1080) cell colonies. We observed that RAD51 clearly promotes extra-chromosomal HR; however, the actions of RAD54 in extra-chromosomal HR were weak. Our data suggest that RAD51 may function as a universal factor during HR, whereas RAD54 mainly functions in other types of HR (gene targeting or intra-chromosomal HR), which involves interaction with chromosomal DNA. 相似文献
7.
Dunlop MH Dray E Zhao W San Filippo J Tsai MS Leung SG Schild D Wiese C Sung P 《The Journal of biological chemistry》2012,287(15):12343-12347
Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair. 相似文献
8.
《DNA Repair》2014
RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1-/- DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy. 相似文献
9.
Lundin C Schultz N Arnaudeau C Mohindra A Hansen LT Helleday T 《Journal of molecular biology》2003,328(3):521-535
The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks. 相似文献
10.
Rice RAD51 paralogs play essential roles in somatic homologous recombination for DNA repair
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Zhan Xu Jianxiang Zhang Meng Xu Wen Ji Meimei Yu Yajun Tao Zhiyun Gong Minghong Gu Hengxiu Yu 《The Plant journal : for cell and molecular biology》2018,95(2):282-295
Synthesis‐dependent strand annealing (SDSA) and single‐strand annealing (SSA) are the two main homologous recombination (HR) pathways in double‐strand break (DSB) repair. The involvement of rice RAD51 paralogs in HR is well known in meiosis, although the molecular mechanism in somatic HR remains obscure. Loss‐of‐function mutants of rad51 paralogs show increased sensitivity to the DSB‐inducer bleomycin, which results in greatly compromised somatic recombination efficiencies (xrcc3 in SDSA, rad51b and xrcc2 in SSA, rad51c and rad51d in both). Using immunostaining, we found that mutations in RAD51 paralogs (XRCC3, RAD51C, or RAD51D) lead to tremendous impairment in RAD51 focus formation at DSBs. Intriguingly, the RAD51C mutation has a strong effect on the protein loading of its partners (XRCC3 and RAD51B) at DSBs, which is similar to the phenomenon observed in the case of blocking PI3K‐like kinases in wild‐type plant. We conclude that the rice CDX3 complex acts in SDSA recombination while the BCDX2 complex acts in SSA recombination in somatic DSB repair. Importantly, RAD51C serves as a fulcrum for the local recruitment of its partners (XRCC3 for SDSA and RAD51B for SSA) and is positively modulated by PI3K‐like kinases to facilitate both the SDSA and SSA pathways in RAD51 paralog‐dependent somatic HR. 相似文献
11.
《DNA Repair》2017
Homologous recombination (HR) serves to repair DNA double-strand breaks and damaged replication forks and is essential for maintaining genome stability and tumor suppression. HR capacity also determines the efficacy of anticancer therapy. Hence, there is an urgent need to better understand all HR proteins and sub-pathways. An emerging protein that is critical for RAD51-mediated HR is RAD51-associated protein 1 (RAD51AP1). Although much has been learned about its biochemical attributes, the precise molecular role of RAD51AP1 in the HR reaction is not yet fully understood. The available literature also suggests that RAD51AP1 expression may be relevant for cancer development and progression. Here, we review the efforts that led to the discovery of RAD51AP1 and elaborate on our current understanding of its biochemical profile and biological function. We also discuss how RAD51AP1 may help to promote cancer development and why it could potentially represent a promising new target for therapeutic intervention. 相似文献
12.
Role of RAD51C and XRCC3 in genetic recombination and DNA repair 总被引:1,自引:0,他引:1
In germ line cells, recombination is required for gene reassortment and proper chromosome segregation at meiosis, whereas in somatic cells it provides an important mechanism for the repair of DNA double-strand breaks. Five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) that share homology with RAD51 recombinase and are known as the RAD51 paralogs are important for recombinational repair, as paralog-defective cell lines exhibit spontaneous chromosomal aberrations, defective DNA repair, and reduced gene targeting. The paralogs form two distinct protein complexes, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3, but their precise cellular roles remain unknown. Here, we show that, like MLH1, RAD51C localized to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, we found that Holliday junction resolvase activity associated tightly and co-eluted with the 80-kDa RAD51C-XRCC3 complex. Taken together, these data indicate that the RAD51C-XRCC3-associated Holliday junction resolvase complex associates with crossovers and may play an essential role in the resolution of recombination intermediates prior to chromosome segregation. 相似文献
13.
TM Kim JH Ko L Hu SA Kim AJ Bishop J Vijg C Montagna P Hasty 《Molecular and cellular biology》2012,32(18):3663-3680
RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements. 相似文献
14.
Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. 相似文献
15.
Stark JM Hu P Pierce AJ Moynahan ME Ellis N Jasin M 《The Journal of biological chemistry》2002,277(23):20185-20194
Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1- and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells. 相似文献
16.
Olivier Da Ines Fabienne Degroote Simon Amiard Chantal Goubely Maria E. Gallego Charles I. White 《The Plant journal : for cell and molecular biology》2013,74(6):959-970
Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51‐DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three ‘non‐meiotic’ paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells. 相似文献
17.
RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis 总被引:1,自引:0,他引:1
Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability. 相似文献
18.
Telomere maintenance requires the RAD51D recombination/repair protein 总被引:12,自引:0,他引:12
The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion. 相似文献
19.
DNA double-strand break repair signalling: the case of RAD51 post-translational regulation 总被引:10,自引:0,他引:10
DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation or by replication block. However, cells can take advantage of DSB-induced recombination in order to generate genetic diversity in physiological processes such as meiosis and V(D)J recombination. Two main alternative pathways compete for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). This review will briefly present the mechanisms and the enzymatic complex for HR and NHEJ. The signalling of the DSB through the ATM pathway will be presented. Then, we will focus on the case of the RAD51 protein, which plays a pivotal role in HR and is conserved from bacteria to humans. Post-translational regulation of RAD51 is presented. Two contrasting situations are discussed: one with up-regulation (expression of the oncogene BCR/ABL) and one with a down-regulation (expression of the oncogene BCL-2) of RAD51, associated with apoptosis inhibition and tumour predisposition. 相似文献