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Ganesh Nagaraju - One of the best experts on this subject based on the ideXlab platform.

  • RAD51C/XRCC3 Facilitates Mitochondrial DNA Replication and Maintains Integrity of the Mitochondrial Genome.
    Molecular and Cellular Biology, 2018
    Co-Authors: Anup Mishra, Sneha Saxena, Anjali Kaushal, Ganesh Nagaraju
    Abstract:

    : Mechanisms underlying mitochondrial genome maintenance have recently gained wide attention, as mutations in mitochondrial DNA (mtDNA) lead to inherited muscular and neurological diseases, which are linked to aging and cancer. It was previously reported that human RAD51, RAD51C, and XRCC3 localize to mitochondria upon oxidative stress and are required for the maintenance of mtDNA stability. Since RAD51 and RAD51 paralogs are spontaneously imported into mitochondria, their precise role in mtDNA maintenance under unperturbed conditions remains elusive. Here, we show that RAD51C/XRCC3 is an additional component of the mitochondrial nucleoid having nucleus-independent roles in mtDNA maintenance. RAD51C/XRCC3 localizes to the mtDNA regulatory regions in the D-loop along with the mitochondrial polymerase POLG, and this recruitment is dependent upon Twinkle helicase. Moreover, upon replication stress, RAD51C and XRCC3 are further enriched at the mtDNA mutation hot spot region D310. Notably, the absence of RAD51C/XRCC3 affects the stability of POLG on mtDNA. As a consequence, RAD51C/XRCC3-deficient cells exhibit reduced mtDNA synthesis and increased lesions in the mitochondrial genome, leading to overall unhealthy mitochondria. Together, these findings lead to the proposal of a mechanism for a direct role of RAD51C/XRCC3 in maintaining mtDNA integrity under replication stress conditions.

  • RAD51C xrcc3 facilitates mitochondrial dna replication and maintains integrity of the mitochondrial genome
    Molecular and Cellular Biology, 2017
    Co-Authors: Anup Mishra, Sneha Saxena, Anjali Kaushal, Ganesh Nagaraju
    Abstract:

    : Mechanisms underlying mitochondrial genome maintenance have recently gained wide attention, as mutations in mitochondrial DNA (mtDNA) lead to inherited muscular and neurological diseases, which are linked to aging and cancer. It was previously reported that human RAD51, RAD51C, and XRCC3 localize to mitochondria upon oxidative stress and are required for the maintenance of mtDNA stability. Since RAD51 and RAD51 paralogs are spontaneously imported into mitochondria, their precise role in mtDNA maintenance under unperturbed conditions remains elusive. Here, we show that RAD51C/XRCC3 is an additional component of the mitochondrial nucleoid having nucleus-independent roles in mtDNA maintenance. RAD51C/XRCC3 localizes to the mtDNA regulatory regions in the D-loop along with the mitochondrial polymerase POLG, and this recruitment is dependent upon Twinkle helicase. Moreover, upon replication stress, RAD51C and XRCC3 are further enriched at the mtDNA mutation hot spot region D310. Notably, the absence of RAD51C/XRCC3 affects the stability of POLG on mtDNA. As a consequence, RAD51C/XRCC3-deficient cells exhibit reduced mtDNA synthesis and increased lesions in the mitochondrial genome, leading to overall unhealthy mitochondria. Together, these findings lead to the proposal of a mechanism for a direct role of RAD51C/XRCC3 in maintaining mtDNA integrity under replication stress conditions.

  • enhanced non homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly adp ribose polymerase
    Carcinogenesis, 2015
    Co-Authors: Kumar Somyajit, Anup Mishra, Aida Jameei, Ganesh Nagaraju
    Abstract:

    Here, we show that PARP inhibitor-mediated cell death of RAD51C-deficient cells occur by NHEJ-driven illegitimate repair of one-ended double-strand breaks, and the hypomorphic RAD51C pathological mutant cells can be targeted by `synergistic toxicity' induced by low-dose PARP inhibitor and IR.Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a `synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G(2)/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity `synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a `synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes.

  • atm and atr mediated phosphorylation of xrcc3 regulates dna double strand break induced checkpoint activation and repair
    Molecular and Cellular Biology, 2013
    Co-Authors: Kumar Somyajit, Ralph Scully, Shivakumar Basavaraju, Ganesh Nagaraju
    Abstract:

    The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

  • distinct roles of fanco RAD51C protein in dna damage signaling and repair implications for fanconi anemia and breast cancer susceptibility
    Journal of Biological Chemistry, 2012
    Co-Authors: Kumar Somyajit, Shreelakshmi Subramanya, Ganesh Nagaraju
    Abstract:

    RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Ralph Scully - One of the best experts on this subject based on the ideXlab platform.

  • atm and atr mediated phosphorylation of xrcc3 regulates dna double strand break induced checkpoint activation and repair
    Molecular and Cellular Biology, 2013
    Co-Authors: Kumar Somyajit, Ralph Scully, Shivakumar Basavaraju, Ganesh Nagaraju
    Abstract:

    The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

  • xrcc2 and xrcc3 regulate the balance between short and long tract gene conversions between sister chromatids
    Molecular and Cellular Biology, 2009
    Co-Authors: Ganesh Nagaraju, Andrea J Hartlerode, Amy Kwok, Gurushankar Chandramouly, Ralph Scully
    Abstract:

    Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the RAD51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/RAD51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.

  • differential regulation of short and long tract gene conversion between sister chromatids by RAD51C
    Molecular and Cellular Biology, 2006
    Co-Authors: Ganesh Nagaraju, Shobu Odate, Ralph Scully
    Abstract:

    The Rad51 paralog RAD51C has been implicated in the control of homologous recombination. To study the role of RAD51C in vivo in mammalian cells, we analyzed short-tract and long-tract gene conversion between sister chromatids in hamster RAD51C / CL-V4B cells in response to a site-specific chromosomal doublestrand break. Gene conversion was inefficient in these cells and was specifically restored by expression of wild-type RAD51C. Surprisingly, gene conversions in CL-V4B cells were biased in favor of long-tract gene conversion, in comparison to controls expressing wild-type RAD51C. These long-tract events were not associated with crossing over between sister chromatids. Analysis of gene conversion tract lengths in CL-V4B cells lacking RAD51C revealed a bimodal frequency distribution, with almost all gene conversions being either less than 1 kb or greater than 3.2 kb in length. These results indicate that RAD51C plays a pivotal role in determining the “choice” between short- and long-tract gene conversion and in suppressing gene amplifications associated with sister chromatid recombination. Double-strand breaks (DSB) are a threat to genome stability, since their misrepair can cause chromosome translocations,

Sergey G Kuznetsov - One of the best experts on this subject based on the ideXlab platform.

  • RAD51C and trp53 double mutant mouse model reveals common features of homologous recombination deficient breast cancers
    Oncogene, 2016
    Co-Authors: Manuela Tumiati, Annabrita Hemmes, Pauliina Munne, Henrik Edgren, S Eldfors, Sergey G Kuznetsov
    Abstract:

    Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, RAD51C, is knocked-out specifically in epidermal tissues. RAD51C is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of RAD51C invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of RAD51C shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of RAD51C-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, RAD51C/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes.

  • loss of RAD51C accelerates tumourigenesis in sebaceous glands of trp53 mutant mice
    The Journal of Pathology, 2015
    Co-Authors: Manuela Tumiati, Annabrita Hemmes, Sanna Uusivirta, Sonja Koopal, Matti Kankainen, Eero Lehtonen, Sergey G Kuznetsov
    Abstract:

    Germline mutations in RAD51C predispose to breast and ovarian cancers. However, the mechanism of RAD51C-mediated carcinogenesis is poorly understood. We previously reported a first-generation RAD51C-knock-out mouse model, in which a spontaneous loss of both RAD51C and Trp53 together resulted in a high incidence of sebaceous carcinomas, particularly in preputial glands. Here we describe a second-generation mouse model, in which RAD51C is deleted, alone or together with Trp53, in sebaceous glands, using Cre-mediated recombination. We demonstrate that deletion of RAD51C alone is not sufficient to drive tumourigenesis and may only cause keratinization of preputial sebocytes. However, deletion of RAD51C together with Trp53 leads to tumour development at around 6 months of age, compared to 11 months for single Trp53-mutant mice. Preputial glands of double-mutant mice are also characterized by increased levels of cell proliferation and DNA damage and form multiple hyperplasias, detectable as early as 2 months of age. Our results reveal a critical synergy between RAD51C and Trp53 in tumour progression and provide a predictable in vivo model system for studying mechanisms of RAD51C-mediated carcinogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  • loss of RAD51C leads to embryonic lethality and modulation of trp53 dependent tumorigenesis in mice
    Cancer Research, 2009
    Co-Authors: Sergey G Kuznetsov, Diana C Haines, Betty K Martin, Shyam K Sharan
    Abstract:

    RecA/Rad51 protein family members (Rad51, Rad51b, RAD51C, Rad51d, Xrcc2, and Xrcc3) are essential for DNA repair by homologous recombination, and their role in cancers has been anticipated. Here we provide the first direct evidence for a tumor suppressor function for a member of the Rad51 family. We show that RAD51C deficiency leads to early embryonic lethality, which can be delayed on a Trp53-null background. To uncover the role of RAD51C in tumorigenesis, we have exploited the fact that RAD51C and Trp53 are both closely located on the mouse chromosome 11. We have generated double heterozygous (DH) mice carrying mutant alleles of both genes either on different (DH-trans) or on the same chromosome (DH-cis), the latter allowing for a deletion of wild-type alleles of both genes by loss of heterozygosity. DH-trans mice, in contrast to DH-cis, developed tumors with latency and spectrum similar to Trp53 heterozygous mice. Strikingly, RAD51C mutation in DH-cis mice promoted the development of tumors of specialized sebaceous glands and suppressed tumors characteristic of Trp53 mutation. In addition, DH-cis females developed tumors significantly earlier than any other group. [Cancer Res 2009;69(3):863–72]

  • resolving RAD51C function in late stages of homologous recombination
    Cell Division, 2007
    Co-Authors: Shyam K Sharan, Sergey G Kuznetsov
    Abstract:

    DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile RAD51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.

  • RAD51C deficiency in mice results in early prophase i arrest in males and sister chromatid separation at metaphase ii in females
    Journal of Cell Biology, 2007
    Co-Authors: Sergey G Kuznetsov, Betty K Martin, Manuela Pellegrini, Kristy Shuda, Oscar Fernandezcapetillo, Sandra Burkett, Eileen Southon, Debananda Pati, Lino Tessarollo, Stephen C West
    Abstract:

    RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of RAD51C in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in RAD51C-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.

David Schild - One of the best experts on this subject based on the ideXlab platform.

  • human RAD51C deficiency destabilizes xrcc3 impairs recombination and radiosensitizes s g2 phase cells
    Journal of Biological Chemistry, 2004
    Co-Authors: David Schild, Mark A Brenneman, Leslie J Redpath, David J Chen
    Abstract:

    Abstract The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, RAD51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, RAD51C was found to be a central component present in two complexes, RAD51C-XRCC3 and Rad51B-RAD51C-Rad51D-XRCC2. We have shown previously that the human RAD51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of RAD51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of RAD51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in RAD51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with RAD51C. In addition, RAD51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of RAD51C-deficient HeLa cells was evident in S and G2/M phases of the cell cycle but not in G1 phase. Together, these results provide direct cellular evidence for the function of human RAD51C in homologous recombinational repair.

  • RAD51C interacts with rad51b and is central to a larger protein complex in vivo exclusive of rad51
    Journal of Biological Chemistry, 2002
    Co-Authors: Kristi A Miller, David Schild, Daniel M Yoshikawa, Ian R Mcconnell, Robin Clark, Joanna S Albala
    Abstract:

    RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.

  • interactions involving the rad51 paralogs RAD51C and xrcc3 in human cells
    Nucleic Acids Research, 2002
    Co-Authors: Claudia Wiese, L. H. Thompson, David W Collins, Joanna S Albala, Amy Kronenberg, David Schild
    Abstract:

    Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, RAD51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and RAD51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or RAD51C. Ni2+-binding experiments demonstrate that XRCC3 and RAD51C interact in human cells. In addition, we find that RAD51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (RAD51CXRCC3 and Rad51B–RAD51C–Rad51D–XRCC2), both containing RAD51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of RAD51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize RAD51C.

  • involvement of RAD51C in two distinct protein complexes of rad51 paralogs in human cells
    Nucleic Acids Research, 2002
    Co-Authors: David Schild, Michael P Thelen, L. H. Thompson
    Abstract:

    Genetic studies in rodent and chicken mutant cell lines have suggested that Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, RAD51C/Rad51L2 and Rad51D/Rad51L3) play important roles in homologous recombinational repair of DNA double-strand breaks and in maintaining chromosome stability. Previous studies using yeast two- and three-hybrid systems have shown interactions among these proteins, but it is not clear whether these interactions occur simultaneously or sequentially in vivo. By utilizing immunoprecipitation with extracts of human cells expressing epitope-tagged Rad51 paralogs, we demonstrate that XRCC2 and Rad51D, while stably interacting with each other, co-precipitate with RAD51C but not with XRCC3. In contrast, RAD51C is pulled down with XRCC3, whereas XRCC2 and Rad51D are not. In addition, Rad51B could be pulled down with RAD51C and Rad51D, but not with XRCC3. These results suggest that RAD51C is involved in two distinct in vivo complexes: Rad51B–RAD51C–Rad51D–XRCC2 and RAD51CXRCC3. In addition, we demonstrate that Rad51 co-precipitates with XRCC3 but not with XRCC2 or Rad51D, suggesting that Rad51 can be present in an XRCC3RAD51C–Rad51 complex. These complexes may act as functional units and serve accessory roles for Rad51 in the presynapsis stage of homologous recombinational repair.

  • mediator function of the human rad51b RAD51C complex in rad51 rpa catalyzed dna strand exchange
    Genes & Development, 2001
    Co-Authors: Stefan Sigurdsson, David Schild, Joanna S Albala, Stephen Van Komen, Wendy Bussen, Patrick Sung
    Abstract:

    Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and RAD51C, are associated in a stable complex. Rad51B–RAD51C complex has ssDNA binding and ssDNA-stimulated ATPase activities. We also examined the functional interaction of Rad51B–RAD51C with Rad51 and RPA. Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency. The competition by RPA for substrate binding can be partially alleviated by Rad51B–RAD51C. This recombination mediator function of Rad51B–RAD51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo.

Joanna S Albala - One of the best experts on this subject based on the ideXlab platform.

  • the atpase motif in rad51d is required for resistance to dna interstrand crosslinking agents and interaction with RAD51C
    Mutagenesis, 2005
    Co-Authors: Aaron M Gruver, Joanna S Albala, Kristi A Miller, Changanamkandath Rajesh, Phillip G Smiraldo, Saravanan Kaliyaperumal, Rachel Balder, Katie M Stiles, Douglas L Pittman
    Abstract:

    Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.

  • The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C.
    Mutagenesis, 2005
    Co-Authors: Aaron M Gruver, Joanna S Albala, Kristi A Miller, Changanamkandath Rajesh, Phillip G Smiraldo, Saravanan Kaliyaperumal, Rachel Balder, Katie M Stiles, Douglas L Pittman
    Abstract:

    Homologous recombination (HR) is a mechanism forrepairing DNA interstrand crosslinks and double-strandbreaks. In mammals, HR requires the activities of theRAD51 family (RAD51, RAD51B, RAD51C, RAD51D,XRCC2, XRCC3 and DMC1), each of which contains con-served ATP binding sequences (Walker Motifs A and B).RAD51D is a DNA-stimulated ATPase that interacts dir-ectlywithRAD51CandXRCC2.Totest thehypothesisthatATP binding and hydrolysis by RAD51D are required forthe repair of interstrand crosslinks, site-directed mutationsin Walker Motif A were generated, and complementationstudies were performed in Rad51d-deficient mouse embry-onic fibroblasts. The K113R and K113A mutants demon-strated arespective 96and 83% decrease in repair capacityrelativetowild-type.Furtherexaminationofthesemutants,by yeast two-hybrid analyses, revealed an 8-fold reductionintheabilitytoassociatewithRAD51Cwhereasinteractionwith XRCC2 was retained at a level similar to the S111Tcontrol. These cell-based studies are the first evidence thatATP binding and hydrolysis by RAD51D are required forefficient HR repair of DNA interstrand crosslinks.IntroductionHomologous recombination (HR) is responsible for suppress-ing the formation of chromosome abnormalities and repairingDNA double-strand breaks and interstrand crosslinks[reviewed in (1–3)]. This high fidelity repair process useshomologous sequence to restore damaged genetic informationand suppress extensive loss of heterozygosity (4). Mammalianproteins involved in HR repair include BRCA1, BRCA2,RAD51 (a RecA homolog) and the RAD51 paralogs(RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 andDMC1). The RAD51 paralogs interact to form at least twostable complexes (a dimer consisting of RAD51CXRCC3and a larger complex containing RAD51B, RAD51C,RAD51D and XRCC2) that assist the RAD51 strand trans-ferase during HR (5–8). With the exception of Dmc1, whichis expressed during gamete formation, disruption of each of theRad51 genes results in increased sensitivity to DNA damageand a genome instability phenotype characterized by variouschromosomal abnormalities including gaps, breaks and trans-locations (9–13). Correlations between altered expression ofHR genes in various malignancies and the increased prevalenceof allelic variants in cancer patients suggest that defects in thisrepair mechanism play a role in carcinogenesis (14–17).Each of the RAD51 paralogs contains two highly conservedATP binding motifs: Motif A (GXXXXGK(T)XXXXXXI/V)and Motif B, (R/KXXXGXXXL) followed by a series ofhydrophobic residues, where X is any amino acid (18). Con-servative substitution of the lysine in Walker Motif A is pre-dicted to result in a protein capable of limited binding, but nothydrolysis, of ATP (19). The loss of a positively chargedresidue at this position will probably abolish ATP binding(20,21). Accordingly, a lysine to arginine substitution in theWalker Motif A of RecA confers cellular sensitivity to DNAdamage and a reduction in genetic recombination withoutaffecting strand exchange activity (22,23). Expression of theanalogous mutant of human RAD51 (K133R) results in adominant inhibitory effect in murine embryonic stem cellscharacterized by increased sensitivity to DNA damagingagents, decreased spontaneous sister-chromatid exchange andreduced HR repair (24). Mutation of the Walker Motifs inRAD51C and XRCC3 results in decreased ability to repairthe DNA interstrand crosslinks introduced by mitomycin C(MMC) (25–27).Not all proteins mediating the RAD51 strand transferaserequire an ability to bind and hydrolyze ATP. Walker Motif Amutants of yeast Rad55 (lysine 49 substituted with arginine oralanine) display increased X-ray sensitivity and sporulationdefects at 23 C, but the identical substitutions in the consensuslysine of Rad57 have no physiological effect (28). Further-more, though XRCC2 is required for the formation of normalRAD51 foci, mutation of Motif A does not affect its role inDNA repair (29). RAD51B binds DNA and displays ATPaseactivity, although mutation of its Walker motifs has not beenreported (30).Understanding the effect of ATP hydrolysis on the assemblyof DNA repair complexes is critical in elucidation of thedouble-strand break repair process (31). Both RecA andRAD51 undergo conformational changes during ATP bindingor hydrolysis that regulate their polymerization on single-stranded DNA (32–35). Evidence is now emerging to suggestthat RAD51-related protein complexes are also controlled bythis small molecule effector. ATP has been shown to stimulatethe binding of RAD51D–XRCC2 to single-stranded DNA (36),and ATP hydrolysis is necessary for normal dynamics of theRAD51CXRCC3 complex (26).

  • nuclear localization of rad51b is independent of RAD51C and brca2
    Mutagenesis, 2005
    Co-Authors: Kristi A Miller, L. H. Thompson, John M. Hinz, Alice N Yamada, Joanna S Albala
    Abstract:

    Human Rad51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. Rad51B (Rad51L1/hRec2) is one of the five known paralogs of human Rad51 found in a multi-protein complex with three other Rad51 paralogs, RAD51C, Rad51D and Xrcc2. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of Rad51B. This is the first report to detail putative interactions of a Rad51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that Rad51B localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of Rad51B, amino acid residues 4-7, mislocalizes Rad51B to the cytoplasm suggesting that this is the nuclear localization signal for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in mammalian cells deficient in RAD51C showed that Rad51B localizes to the nucleus independent of RAD51C; further suggesting that Rad51B, like RAD51C, contains its own nuclear localization signal.

  • RAD51C interacts with rad51b and is central to a larger protein complex in vivo exclusive of rad51
    Journal of Biological Chemistry, 2002
    Co-Authors: Kristi A Miller, David Schild, Daniel M Yoshikawa, Ian R Mcconnell, Robin Clark, Joanna S Albala
    Abstract:

    RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.

  • interactions involving the rad51 paralogs RAD51C and xrcc3 in human cells
    Nucleic Acids Research, 2002
    Co-Authors: Claudia Wiese, L. H. Thompson, David W Collins, Joanna S Albala, Amy Kronenberg, David Schild
    Abstract:

    Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, RAD51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and RAD51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or RAD51C. Ni2+-binding experiments demonstrate that XRCC3 and RAD51C interact in human cells. In addition, we find that RAD51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (RAD51CXRCC3 and Rad51B–RAD51C–Rad51D–XRCC2), both containing RAD51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of RAD51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize RAD51C.