Mediator function of the human Rad51B–Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange

Sigurdsson, Stefan; Komen, Stephen Van; Bussen, Wendy; Schild, David; Albala, Joanna S.; Sung, Patrick

doi:10.1101/gad.935501

Cited by 214 publications

(194 citation statements)

References 33 publications

Supporting

Mentioning

178

Contrasting

Unclassified

Order By: Relevance

“…DISCUSSION Using multiple methods (both in vitro and in vivo) we have shown that RAD51B and RAD51C proteins form a heterocomplex. These studies are consistent with the results of Schild et al (8,18) and Sung (26), indicating an interaction between RAD51B and RAD51C. Unique RAD51B and RAD51C baculoviruses were generated and co-infected into insect cells to pro- duce recombinant proteins, which were co-purified and characterized using RAD51B-and RAD51C-specific antibodies.…”

Section: Rad51c Interacts With Rad51b Within a Larger Complex In Vivosupporting

confidence: 74%

“…This complex has been shown to perform homologous pairing in the absence of RAD51 (20). In a recent study the RAD51B/RAD51C proteins have been shown to participate in recombinational repair processes similar to the RAD55-RAD57 complex in vitro (26). This would suggest that RAD51C has multiple roles in vivo, pairwise with XRCC3 and RAD51B, or in a larger complex with the other RAD51 paralogs possibly dependent on specific cell type (as has been suggested for the XRCC3/RAD51C complex in brain).…”

Section: Rad51c Interacts With Rad51b Within a Larger Complex In Vivomentioning

confidence: 99%

See 1 more Smart Citation

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

Miller¹,

Yoshikawa²,

McConnell³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.The human RAD51 protein functions in homologous recombination and DNA double-strand break repair (1-4). Five paralogs of human RAD51 have been identified: RAD51B (hREC2, RAD51L1), RAD51C (RAD51L2), RAD51D (RAD51L3), XRCC2, and XRCC3; these proteins share ϳ25% amino acid sequence identity with one another and RAD51 (5-9). DT40 chicken cell knockouts have been generated for each paralog, all of which exhibit a lack of RAD51 foci formation as well as enhanced radiation and cisplatin sensitivity, consistent with a deficiency in recombinational repair (10, 11). These results are consistent with previous work, which showed that the XRCC3 knockout CHO cell line (irs1SF) is deficient in RAD51 foci formation, suggesting that XRCC3 is required for RAD51 function (12). Similarly, XRCC2-defective cell lines also fail to form damage-dependent RAD51 foci (13). Furthermore, both XRCC2 and XRCC3 have been shown to be required for repair of double-strand breaks by homologous recombination in vivo (14 -16). Initially by sequence and now by association and function, it has become increasingly evident that these RAD51 paralogs participate together in recombination and repair processes.Extensive yeast two-and three-hybrid analysis suggests that there are a variety of putative protein-protein interactions between the RAD51 paralogs. These include XRCC2/RAD51D (17, 18) and XRCC3/RAD51(19). Moreover, interactions have been suggested between RAD51C/RAD51B, RAD51C/RAD51D, RAD51C/XRCC3, and RAD51C/RAD51 (8, 18). More recent biochemical evidence has corroborated the interaction between XRCC2 and RAD51D by co-purification of the recombinant proteins and co-elution of the native proteins by gel filtration from mammalian cell extracts; RAD51D was further shown to be a DNA-stimulated ATPase (17). In addition, a stable heterocomplex was demons...

show abstract

Section: Rad51c Interacts With Rad51b Within a Larger Complex In Vivosupporting

confidence: 74%

Section: Rad51c Interacts With Rad51b Within a Larger Complex In Vivomentioning

confidence: 99%

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

Miller¹,

Yoshikawa²,

McConnell³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This result has important biological implications demonstrating that RecA/RAD51 BM of HJs can occur in the cell, where SSB/RPA is abundant. This result is also consistent with a mechanism of BM that involves polymerization of RecA/RAD51 on the displaced ssDNA strand; it was shown that under similar conditions RecA and RAD51 can displace SSB/RPA from ssDNA, when SSB/RPA is added to ssDNA prior or at the same time as RecA/RAD51 (29,30).…”

Section: Resultssupporting

confidence: 74%

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

Rossi

Mazina

Bugreev

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.H omologous recombination (HR) is the process responsible for maintaining genome stability in all living organisms; it is particularly important for repairing DNA double-strand breaks (1-5). The process of HR involves the enzymatic degradation of broken dsDNA ends into resected DNA duplex with protruding ssDNA tails (6, 7). Following resection, the central protein of HR, the prokaryotic (bacterial recombinase protein) RecA or its eukaryotic homolog Rad51, is loaded onto the ssDNA tails and forms a contiguous nucleoprotein filament (8, 9). Although RecA and Rad51 share only ∼30% sequence homology, the filaments they form and the conformational changes they induce in DNA are nearly identical (10).The RecA/Rad51-ssDNA filament possesses the unique ability to search for homologous dsDNA sequences and promote DNA strand exchange: an invasion of ssDNA into homologous DNA duplex that results in the displacement of the identical ssDNA from the duplex and formation of a joint molecule (JM) ( Fig. S1A) (11). Besides its essential DNA strand-exchange activity RecA/RAD51 recombinases have an ability to extend JMs by a kinetically distinct process known as heteroduplex extension or three-strand branch migration (BM), in which one DNA strand is progressively exchanged for another (11,12). When the extending heteroduplex reaches the ssDNA-dsDNA junction on the invading DNA strand, the 3-stranded JM is converted into a 4-stranded Holliday junction (HJ). Then, specialized DNA translocating proteins, like Escherichia coli RuvAB, bind to HJs and promote their migration by four-strand BM (otherwise referred to as the BM of HJs) (13). These DNA translocases are capable of bypassing sequence heterologies and disrupting nucleoprotein complexes encountered during BM. Finally, DNA repair synthesis by DNA polymerases takes place on JMs followed by their resolution.Surprisingly, the RecA/Rad51 recombinases can also promote four-strand BM (Fig. S1A) (14, 15). This activity may play a significant role in the initial stages of recombination in vivo by helping to form and stabilize HJs, and therefore it is important to u...

show abstract

“…During the course of our research, the Rad51B⅐Rad51C complex was purified in the Sung laboratory (46). They reported that the Rad51B⅐Rad51C complex acts to facilitate the replacement of RPA from the nucleofilaments by Rad51 and to promote the DNA strand exchange activity of Rad51 (46), and they suggest a mediator role for the Rad51B⅐Rad51C complex in HRR. We found, using oligonucleotide DNA substrates, that Rad51C, but not Rad51B, promotes a DNA strand exchange reaction.…”

Section: Discussionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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...

show abstract

Mediator function of the human Rad51B–Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange

Cited by 214 publications

References 33 publications

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

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

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

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

Contact Info

Product

Resources

About