Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro

Lio, Yi Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

doi:10.1074/jbc.m211038200

Cited by 112 publications

(115 citation statements)

References 49 publications

(65 reference statements)

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“…In these complexes, Rad51C interacts directly with XRCC3, Rad51B, and Rad51D. Rad51C was also shown to weakly interact with Rad51 in vitro (23,24). The previous protein-protein interaction data have suggested a central role for Rad51C among the five paralogs.…”

Section: Rad51c Dynamically Influences the Protein Levels Of Other Rad51mentioning

confidence: 91%

“…Antibodies-Rad51B and Rad51C antibodies were generated as described previously (24). Polyclonal antiserum against human Rad51D was raised in rabbits using a synthetic peptide (CGTWGTSEQS-ATLQGDQT, Zymed Lab) as immunogen.…”

Section: Methodsmentioning

confidence: 99%

“…To determine the functions of Rad51C in human cells, we have used previously in vitro assays to demonstrate that the purified human Rad51C protein exhibits DNA binding, ATPase, and double-stranded DNA separation activities (24). These findings underscore the potential significance of the human Rad51C in the DNA strand exchange events of HR.…”

mentioning

confidence: 98%

“…In human cells, Rad51C participates in various paralog complexes, including Rad51B-Rad51C, Rad51C-XRCC3, and Rad51B-Rad51C-Rad51D-XRCC2 (18 -22). In terms of proteinprotein interactions, Rad51C apparently has a central role, interacting directly with Rad51B, Rad51D, and XRCC3 and also weakly with Rad51 (23,24). However, the functional significance of these complexes is not yet clear.…”

mentioning

confidence: 99%

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Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Lio

Schild

Brenneman

et al. 2004

Journal of Biological Chemistry

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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 G 2 /M phases of the cell cycle but not in G 1 phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.In mammalian cells, DNA double strand breaks (DSBs) 1 are repaired primarily by two distinct mechanisms, non-homologous end joining (NHEJ), a non-templated, potentially errorprone process in which nucleotide alternations are tolerated at the site of rejoining, and homologous recombination (HR), a largely error-free process in which a sister chromatid or homologous chromosome is used as a template for repair (for review, see Refs. 1 and 2). Homologous recombinational repair (HRR) provides high fidelity in repairing DNA damage and is therefore essential and critical for the maintenance of genome stability and tumor avoidance (for review, see Refs. 3 and 4). The Rad51 protein plays a key role in HR, functioning to mediate homologous DNA pairing and strand exchange (5, 6). Five vertebrate Rad51 paralogs are expressed in mitotically growing cells: Rad51B (7-9), Rad51C (10), Rad51D (9, 11, 12), XRCC2 (13-15), and XRCC3 (13,16,17). These proteins share 20 -30% sequence identity with Rad51 and with each other. Only vertebrates appear to contain all five of these Rad51 paralogs. In human cells, Rad51C participates in various paralog complexes, including Rad51B-Rad51C, Rad51C-XRCC3, and Rad51B-Rad51C-Rad51D-XRCC2 (18 -22). In terms of proteinprotein interactions, Rad51C apparently has a central role, interacting directly with Rad51B, Rad51D, and XRCC3 and also weakly with Rad51 (23,24). However, the functional significance of these complexes is not yet clear.Mutant studies provide a direct means for identifying the function of genes. A knock-out ...

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Section: Rad51c Dynamically Influences the Protein Levels Of Other Rad51mentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

See 2 more Smart Citations

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Lio

Schild

Brenneman

et al. 2004

Journal of Biological Chemistry

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“…When Rad51C retained its ability to bind ATP, it was able to partially restore MMC resistance in irs3 mutant V79 cells, suggesting that ATP binding, rather than hydrolysis, may be critical for Rad51C function. Rad51B also hydrolyzes ATP, and the Rad51C-Rad51B dimer exhibited additive ATPase activity, although the biological significance of the Rad51B ATPase activity is unknown (5,22).…”

Section: From the Biology And Biotechnology Research Program Lawrencmentioning

confidence: 99%

XRCC3 ATPase Activity Is Required for Normal XRCC3-Rad51C Complex Dynamics and Homologous Recombination

Yamada

Hinz

Kopf

et al. 2004

Journal of Biological Chemistry

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Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His 6 -tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mM ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.Homologous recombinational repair (HRR) 1 is a major DNA repair pathway that preserves chromosomal integrity during DNA replication and contributes to the removal of doublestrand breaks and interstrand cross-links from exogenous agents (see reviews in Refs. 1-3). HRR probably helps prevent double-strand breaks from arising during normal DNA replication and promotes their removal in an error-free manner when they do arise. In eukaryotic cells, this process is mediated by the highly conserved Rad51 DNA strand transferase and associated proteins that include distant relatives of Rad51, which are referred to as the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D). At least two stable complexes (a dimeric complex composed of XRCC3 and Rad51C and a larger complex composed of XRCC2, Rad51B, Rad51C, and Rad51D) have been found (4 -8). However, the exact role(s) these complexes play in HRR remains unclear. Mutations in the paralog genes lead to excessive spontaneous chromosomal aberrations and sensitivity to ionizing radiation (IR) and DNA cross-links (9 -13).Rad51 contains ATPase motifs composed of the Walker A and B boxes, and its DNA binding (14) and strand transferase activities (15, 16) are ATP-dependent. Structural analysis of archeal Rad5...

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Human cell toxicogenomic analysis of bromoacetic acid: A regulated drinking water disinfection by‐product

Muellner

Attene‐Ramos

Hudson

et al. 2009

Environ and Mol Mutagen

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The disinfection of drinking water is a major achievement in protecting the public health. However, current disinfection methods also generate disinfection by-products (DBPs). Many DBPs are cytotoxic, genotoxic, teratogenic, and carcinogenic and represent an important class of environmentally hazardous chemicals that may carry long-term human health implications. The objective of this research was to integrate in vitro toxicology with focused toxicogenomic analysis of the regulated DBP, bromoacetic acid (BAA) and to evaluate modulation of gene expression involved in DNA damage/repair and toxic responses, with nontransformed human cells. We generated transcriptome profiles for 168 genes with 30 min and 4 hr exposure times that did not induce acute cytotoxicity. Using qRT-PCR gene arrays, the levels of 25 transcripts were modulated to a statistically significant degree in response to a 30 min treatment with BAA (16 transcripts upregulated and nine downregulated). The largest changes were observed for RAD9A and BRCA1. The majority of the altered transcript profiles are genes involved in DNA repair, especially the repair of double strand DNA breaks, and in cell cycle regulation. With 4 hr of treatment the expression of 28 genes was modulated (12 upregulated and 16 downregulated); the largest fold changes were in HMOX1 and FMO1. This work represents the first nontransformed human cell toxicogenomic study with a regulated drinking water disinfection by-product. These data implicate double strand DNA breaks as a feature of BAA exposure. Future toxicogenomic studies of DBPs will further strengthen our limited knowledge in this growing area of drinking water research.

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Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro

Cited by 112 publications

References 49 publications

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

Human Rad51C Deficiency Destabilizes XRCC3, Impairs Recombination, and Radiosensitizes S/G2-phase Cells

XRCC3 ATPase Activity Is Required for Normal XRCC3-Rad51C Complex Dynamics and Homologous Recombination

Human cell toxicogenomic analysis of bromoacetic acid: A regulated drinking water disinfection by‐product

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