Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2

Shin, David

doi:10.1093/emboj/cdg429

Cited by 248 publications

(321 citation statements)

References 42 publications

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“…Such disorder of the N-terminal domain already has been described for an archaeal Sulfolobus solfataricus RadA filament (a homolog of RAD51), where this domain was visualized in filaments prepared with adenosine 5Ј-[␥-thio]triphosphate but absent in filaments prepared with ATP and aluminum fluoride (18). In addition, a similar disordering of the N-terminal domain was seen in a crystal structure of the archaeal Pyrococcus furiosus RadA protein (PfRad51), where in a heptameric ring only one of the seven N-terminal domains was resolved (19). In a crystal structure of the homologous human Dmc1, no N-terminal domains are seen within an octameric ring of this protein because of substantial disorder (20).…”

Section: Resultsmentioning

confidence: 72%

BRCA2 BRC motifs bind RAD51–DNA filaments

Galkin

Esashi

Xiong

et al. 2005

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

127

118

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Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.electron microscopy ͉ nucleoprotein filaments ͉ recombination ͉ genome stability and dynamics ͉ structural biology M utations in BRCA2 are associated with an increased risk of breast and ovarian cancers, and almost half the cases of inherited early onset breast cancers have been linked to mutations in BRCA2. The product of this gene, BRCA2 protein (384 kDa), interacts with RAD51, and it has been shown that both proteins are essential for homologous recombination, DNA repair, and the maintenance of genome stability. There appear to be a number of different interactions between BRCA2 and RAD51. One such interaction involves a C-terminal region of BRCA2 (1). In addition, BRCA2 interacts with RAD51 through the eight conserved BRC repeats in BRCA2 (2, 3), and mutations within these repeats are associated with cancer predisposition. The deletion of several BRC repeats in mice leads to cancer (4), and somatic mutations in BRC repeats have been found to be associated with breast cancer (5). Although there are eight BRC repeats in human BRCA2, in other organisms the number of BRC repeats is quite variable, with 15 found in a Trypanosoma BRCA2-like protein (6) and only one found in the Ustilago Brh2 protein (7).Many details of the interactions between BRCA2 and RAD51 are unclear, such as whether BRCA2 binds to the N-terminal domain of RAD51 (8) or to the nucleotide-binding core (3, 9). Recently, a fusion protein containing the nucleotide-binding core of RAD51 and a single BRC repeat was crystallized. Analysis of the x-ray structure of the fusion led t...

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Section: Resultsmentioning

confidence: 72%

BRCA2 BRC motifs bind RAD51–DNA filaments

Galkin

Esashi

Xiong

et al. 2005

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

127

118

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“…This domain is very similar in DMC1 and RAD51, whereas the N-terminal regions of the two recombinases are more divergent. Recombinant RAD51 and DMC1 proteins form oligomeric ring structures of seven or eight monomers, respectively (Shin et al, 2003;Kinebuchi et al, 2004). However, they are functionally active when associated with DNA in the form of a highly ordered right-handed helical nucleoprotein filament (Figure 3b) (Benson et al, 1994;Conway et al, 2004;Sehorn et al, 2004).…”

Section: Predominant In G1mentioning

confidence: 99%

BRCA2: a universal recombinase regulator

2007

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Homologous recombination has a dual role in eukaryotic organisms. Firstly, it is responsible for the creation of genetic variability during meiosis by directing the formation of reciprocal crossovers that result in random combinations of alleles and traits. Secondly, in mitotic cells, it maintains the integrity of the genome by promoting the faithful repair of DNA double-strand breaks (DSBs). In vertebrates, it therefore plays a key role in tumour avoidance. Mutations in the tumour suppressor protein BRCA2 are associated with predisposition to breast and ovarian cancers, and loss of BRCA2 function leads to genetic instability. BRCA2 protein interacts directly with the RAD51 recombinase and regulates recombinationmediated DSB repair, accounting for the high levels of spontaneous chromosomal aberrations seen in BRCA2-defective cells. Recent observations indicate that BRCA2 also plays a critical role in meiotic recombination, this time through direct interactions with the meiosis-specific recombinase DMC1. The interactions of BRCA2 with RAD51 and DMC1 lead us to suggest that the BRCA2 tumour suppressor is a universal regulator of recombinase actions.

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“…Structural analysis of archeal Rad51 shows that the ATPase binding site is composed of Walker A and Walker B motifs from different Rad51 monomers (17), suggesting that ATP binding may act as a "bridge" for protein-protein interaction. Interestingly, a conservative lysine to arginine mutation in the Walker A box resulted in a mutant human Rad51 with efficient DNA binding and strand transferase activity while a nonconservative lysine to alanine mutation inactivated Rad51 (18).…”

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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Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2

Cited by 248 publications

References 42 publications

BRCA2 BRC motifs bind RAD51–DNA filaments

BRCA2 BRC motifs bind RAD51–DNA filaments

BRCA2: a universal recombinase regulator

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

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