Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer
Introduction
DNA double-strand breaks (DSB) occur frequently in dividing cells, and erroneous repair of these lesions can predispose to cancer and other human diseases. In many organisms, homologous recombination (HR) provides a pathway for repairing DSBs with high fidelity [1], [2]. In humans the execution of this pathway requires the RAD51 recombinase and a host of accessory proteins, including RAD50, RAD54, MRE11, NBS1 and replication protein A (RPA). Five RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, and the breast cancer susceptibility proteins, BRCA1 and BRCA2, have also been implicated in HR, although their precise functions are unclear [3], [4]. Deficiencies of these genes in vertebrate cell lines lead invariably to greater sensitivity to genotoxic agents, albeit to different extents [5], [6], [7], [8]. In particular, germline mutations in the human BRCA2 gene have been reported to confer increased susceptibility to breast, ovarian, prostate, and pancreatic cancers, and may cause Fanconi’s anaemia [9], [10], [11]. Clearly, a better understanding of the molecular and functional complexities of these proteins is necessary to unlock the underlying causes of such hereditary diseases.
Composed of two globular domains, RAD51 protein catalyses ATP-dependent homologous pairing and strand exchange between DNA sister chromatids or possibly homologous chromosomes. RAD51 multimerises, in a polar and helical manner, to form an ordered filament that sheathes the 3′-single-stranded ends of resected DSBs [12], the resulting filamentous structure being the active state to conduct genetic recombination. The RAD51 C-terminal domain (amino acids (aa) 98–339), which is homologous to the core domain of Escherichia coli RecA [13], is important for multimerisation and accounts for much of the catalytic property; the N-terminal domain (aa 1–82) protrudes from the filament and forms lobes that perhaps interact with DNA [14]. The cellular functioning of RAD51 requires interplay with other signalling and helper proteins, and both its role as a tumour suppressor and its ability to bind RAD51 in vivo have suggested that BRCA2 may be one such player [8], [15], [16].
Human BRCA2 is a multi-domain protein with 3418 aa. Like other DNA repair proteins it is localised to the cell nucleus [17]. Its lack of homology to proteins of known function has hampered a priori functional analysis in the past. Crystallographic studies, however, have revealed that the C-terminal region of BRCA2 contains DNA/DSS1-binding domains (DBD) (aa 2478–3185), which comprise the helical domain, OB1, OB2, the Tower region, and OB3 [18]. The helical domain and OB1 together interact stably with DSS1, a protein deleted in split-hand split-foot syndrome [19], [20]. Single-stranded DNA (ssDNA) binding has been attributed to two of the three oligonucleotide/oligosaccharide-binding (OB) folds—also found in the ssDNA-binding protein RPA [21]—whereas the Tower region has been implicated in interaction with double-stranded DNA (dsDNA) [18]. In addition to DBD, BRCA2 has a large central exon 11, which contains eight similar sequence motifs, called BRC repeats [22]. Each of the eight BRC repeats (BRC1–BRC8) is composed of about 30 aa. Yeast two hybrid and immunoprecipitation studies have provided evidence for direct interactions between a number of BRC repeats and RAD51 [15], [16], [23]. The 1.7 Å resolution crystal structure of RAD51 C-terminal domain, complexed with BRC4 [24], shows that the RAD51 C-terminal domain forms a mixed α–β-fold (Fig. 2A), with two disordered loops, L1 (aa 230–236) and L2 (aa 269–292), which have been suggested as potential DNA-binding sites [25], [26]. BRC4 extends the RAD51 core β-pleated sheet by two short strands, and packs an amphipathic α-helix (aa 1536–1542) against the RAD51 exterior. A strip of hydrophobic contacts, as well as two main points of charge–charge interaction, is found in the interface.
The structure of the BRC4–RAD51 complex provides a useful model for the interactions between homologues of the BRC repeat and RAD51. Critical residues for interaction may be picked out in the form of a sequence fingerprint, and be compared within and across species. We describe the eukaryotic BRCA2 homologues, and present an analysis of the binding capabilities of a diverse range of BRC repeat and RAD51 homologues. We also discuss the possible implications of our results to BRCA2 function, DNA recombination and DNA repair.
Section snippets
Defining interaction sequence fingerprints for the BRC repeat and RAD51
Sequence fingerprints representing key residues for BRCA2–RAD51 interactions were derived from a detailed analysis of the BRC4–RAD51 interface (Protein Data Bank (PDB) code: 1n0w). For each of the two subunits, we selected residues with atoms within 5 Å of the other subunit. We then examined residue solvent accessibilities, in bound and unbound states, and the nature of the residue contacts in the interface, using residue environment annotations, generated by the program JOY [27], to facilitate
Identification and comparisons of BRCA2-like proteins
Proteins with full or partial homology to human BRCA2 exist in a diverse range of eukaryotes, from plants to parasitic organisms [22], [35], [36]. Evidence that a partial BRCA2 homologue (Brh2) is required for maintaining genome stability in the fungus Ustilago maydis has implicated a general role for BRCA2-like proteins in DNA repair among eukaryotic organisms [36]. Using the sequence profile-based method PSI-BLAST [29] and the pattern-based PHI-BLAST, we identified full and partial homologues
Discussion
BRCA2 homologues with BRC repeat motifs have been identified in a diverse range of eukaryotic organisms. Using the structure-based sequence fingerprints, we have observed conservation of key interaction residues in eukaryotic BRC repeats and RAD51 orthologues, and thus predict that BRC repeat–RAD51 interactions occur in many eukaryotic species, though with important exceptions in some fungi. Remarkably, the biologically well-characterised S. cerevisiae and S. pombe do not appear to have such
Acknowledgements
We thank Andy Dore, Ben Luisi, and Kenji Mizuguchi for helpful advice and discussions. This research was funded by the Biological and Biotechnological Science Research Council and the Wellcome Trust.
References (60)
- et al.
Genomic integrity and the repair of double-strand DNA breaks
Mutat. Res.
(2001) Cancer susceptibility and the functions of BRCA1 and BRCA2
Cell
(2002)Involvement of Brca2 in DNA repair
Mol. Cell
(1998)The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR
J. Mol. Biol.
(1999)RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene brca2
J. Biol. Chem.
(1997)Basic local alignment search tool
J. Mol. Biol.
(1990)- et al.
A knowledge base for predicting protein localization sites in eukaryotic cells
Genomics
(1992) - et al.
Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming
J. Mol. Biol.
(1990) - et al.
FUGUE: sequence–structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties
J. Mol. Biol.
(2001) - et al.
Comparative protein modelling by satisfaction of spatial restraints
J. Mol. Biol.
(1993)
BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis
Mol. Cell
Role of BRCA2 in control of the RAD51 recombination and DNA repair protein
Mol. Cell
Expression of BRC repeats in breast cancer cells disrupts the BRCA2–Rad51 complex and leads to radiation hypersensitivity and loss of G(2)/M checkpoint control
J. Biol. Chem.
Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation
J. Biol. Chem.
Specific interactions between the human RAD51 and RAD52 proteins
J. Biol. Chem.
Visualisation of human rad52 protein and its complexes with hRad51 and DNA
J. Mol. Biol.
Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA
J. Mol. Biol.
Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells
J. Biol. Chem.
Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase
J. Biol. Chem.
XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination
Mol. Cell
XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages
Mol. Cell
Xrcc3 is required for assembly of Rad51 complexes in vivo
J. Biol. Chem.
Homologous DNA recombination in vertebrate cells
Proc. Natl. Acad. Sci. U.S.A.
Mammalian recombination–repair genes XRCC2 and XRCC3 promote correct chromosome segregation
Nat. Cell. Biol.
The Rad51 paralog Rad51B promotes homologous recombinational repair
Mol. Cell. Biol.
Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs
Mol. Cell. Biol.
The genetics of breast cancer susceptibility
Annu. Rev. Genet.
Breast cancer genetics: what we know and what we need
Nat. Med.
Biallelic inactivation of BRCA2 in Fanconi anemia
Science
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