RAD51C Interacts with RAD51B and Is Central to a Larger Protein Complex in Vivo Exclusive of RAD51

Miller, Kristi A.; Yoshikawa, Daniel M.; McConnell, Ian R.; Clark, Robin; Schild, David; Albala, Joanna S.

doi:10.1074/jbc.m108306200

Cited by 81 publications

(66 citation statements)

References 25 publications

(43 reference statements)

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“…This is the first in vitro evidence for Rad51⅐Rad51C⅐Rad51B complex formation. However, in the course of our study, it was reported that overexpressed His 6 -tagged Rad51C did not pull down Rad51 in human cells (43,44). Because we observed that the interaction between Rad51C and Rad51 is weak, only ϳ5-10% of the overexpressed Rad51 was pulled down by the Rad51B⅐Rad51C complex in insect cell extracts as well as by purified complex of Rad51B and Rad51C proteins, the failure to detect the Rad51C-Rad51 interaction in human cells (43,44) is likely due to the weak interaction between the Rad51B⅐Rad51C complex and Rad51.…”

Section: Discussionmentioning

confidence: 71%

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

Lio¹,

Mazin²,

Kowalczykowski³

et al. 2003

Journal of Biological Chemistry

112

109

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The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/ Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His) 6 , and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B⅐Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single-and double-stranded DNA and to preferentially bind 3-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway. Homologous recombinational repair (HRR)1 is an important pathway in repairing DNA double-strand breaks (DSBs) with high accuracy, which is indispensable for the maintenance of genome stability (for review see Refs. 1-4). The human Rad51 protein, a structural and functional homolog of Escherichia coli recombinase RecA (5, 6), plays a central role in HRR. In vitro studies have shown that Rad51 binds single-stranded and double-stranded DNA (ssDNA and dsDNA), exhibits DNA-dependent ATPase activity, and functions to catalyze homologous pairing and DNA strand exchange (6 -8). As a prerequisite step for the action of Rad51 in HRR, Rad51 binds DNA to form highly ordered nucleoprotein filaments (9, 10). It has also been shown that these filaments form preferentially on tailed duplex DNA substrates that mimic the 3Ј-overhanging ssDNA tails at the sites of DSBs (11). Thus far, Rad51 is the only mitotic protein that catalyzes the key reactions of homologous pairing and strand transfer.In yeast, the Rad51-related proteins include Rad55 and Rad57, which exist together as a tight heterodimer that interacts weakly with Rad51 (12). These two proteins appear to be Rad51 paralogs, probably derived by gene duplication followed by the evolution of new functions. The Rad55⅐Rad57 complex acts as a cofactor for the assembly of Rad51 onto ssDNA and functions to promote the DNA strand transfer activity of Rad51 (12). In human cells, in addition to meiotic Dmc1 protein, five Rad51 paralogs have been identified: Rad51B/Rad51L1 2 (13-15), Rad51C/Rad51L2 (16), Rad51D/Rad51L3 (15, 17, 18), XRCC2 (19 -21), and XRCC3 (19,22,23). These Rad51 paralogs share 20 -30% sequence id...

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Section: Discussionmentioning

confidence: 71%

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

Lio¹,

Mazin²,

Kowalczykowski³

et al. 2003

Journal of Biological Chemistry

112

109

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“…In the absence of EME1, chromosomal aberrations occurred and led to genomic instability (Abraham et al, 2003). The gene RAD51L1 (fc: 11.395, DON2.5) functions in homologous recombination and DNA double-strand break repair (Miller et al, 2002). CHAF1B (fc: 1 1.435, DON5) is part of the complex CAF-1 and the only subunit available on the Chicken Genome Array.…”

Section: Dna Repairmentioning

confidence: 99%

Fusarium mycotoxin-contaminated wheat containing deoxynivalenol alters the gene expression in the liver and the jejunum of broilers

Dietrich

Neuenschwander

Bucher

et al. 2012

Animal

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The effects of mycotoxins in the production of animal feed were investigated using broiler chickens. For the feeding trial, naturally Fusarium mycotoxin-contaminated wheat was used, which mainly contained deoxynivalenol (DON). The main effects of DON are reduction of the feed intake and reduced weight gain of broilers. At the molecular level, DON binds to the 60 S ribosomal subunit and subsequently inhibits protein synthesis at the translational level. However, little is known about other effects of DON, for example, at the transcriptional level. Therefore, a microarray analysis was performed, which allows the investigation of thousands of transcripts in one experiment. In the experiment, 20 broilers were separated into four groups of five broilers each at day 1 after hatching. The diets consisted of a control diet and three diets with calculated, moderate concentrations of 1.0, 2.5 and 5.0 mg DON/kg feed, which was attained by exchanging uncontaminated wheat with naturally mycotoxin-contaminated wheat up to the intended DON concentration. The broilers were held at standard conditions for 23 days. Three microarrays were used per group to determine the significant alterations of the gene expression in the liver (P , 0.05), and qPCR was performed on the liver and the jejunum to verify the results. No significant difference in BW, feed intake or feed conversion rate was observed. The nutrient uptake into the hepatic and jejunal cells seemed to be influenced by genes: SLC2A5 (fc: 21.54, DON2.5), which facilitates glucose and fructose transport and SLC7A10 (fc: 11.49, DON5), a transporter of D-serine and other neutral amino acids. In the jejunum, the palmitate transport might be altered by SLC27A4 (fc: 21.87, DON5) and monocarboxylates uptake by SLC16A1 (fc: 21.47, DON5). The alterations of the SLC gene expression may explain the reduced weight gain of broilers chronically exposed to DON-contaminated wheat. The decreased expressions of EIF2AK3 (fc: 21.29, DON2.5/5) and DNAJC3 (fc: 21.44, DON2.5) seem to be related to the translation inhibition. The binding of DON to the 60 S ribosomal subunit and the subsequent translation inhibition might be counterbalanced by the downregulation of EIF2AK3 and DNAJC3. The genes PARP1, MPG, EME1, XPAC, RIF1 and CHAF1B are mainly related to single-strand DNA modifications and showed an increased expression in the group with 5 mg DON/kg feed. The results indicate that significantly altered gene expression was already occurring at 2.5 mg DON/kg feed.

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“…a heterodimer of Rad51C and XRCC3 and a larger complex comprising Rad51B, Rad51C, Rad51D, and XRCC2 (18, 20 -22). The transient existence of Rad51B-Rad51C and Rad51D-XRCC3 heterodimers also has been suggested (21,22,56). It seems probable that the various paralog complexes form dynamically during the process of HR (21).…”

Section: Rad51c Dynamically Influences the Protein Levels Of Other Rad51mentioning

confidence: 99%

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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RAD51C Interacts with RAD51B and Is Central to a Larger Protein Complex in Vivo Exclusive of RAD51

Cited by 81 publications

References 25 publications

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

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

Fusarium mycotoxin-contaminated wheat containing deoxynivalenol alters the gene expression in the liver and the jejunum of broilers

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

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