Mutational analysis of Brh2 reveals requirements for compensating mediator functions

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.     

The BRCA2-interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis

DSS1 encodes a small acidic protein shown in recent structural studies to interact with the DNA binding domain of BRCA2. Here we report that an ortholog of DSS1 is present in Ustilago maydis and associates with Brh2, the BRCA2-related protein, thus recapitulating the protein partnership in this genetically amenable fungus. Mutants of U. maydis deleted of DSS1 are extremely radiation sensitive, deficient in recombination, defective in meiosis, and disturbed in genome stability; these phenotypes mirror previous observations of U. maydis mutants deficient in Brh2 or Rad51. These findings conclusively show that Dss1 constitutes a protein with a significant role in the recombinational repair pathway in U. maydis, and imply that it plays a similar key role in the recombination systems of organisms in which recombinational repair is BRCA2 dependent.     

Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis

Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system.     

Requirement of mouse BCCIP for neural development and progenitor proliferation

Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR) pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors.     

Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress

Mutants of the fungus Ustilago maydis defective in the RecQ helicase Blm are highly sensitive to killing by the DNA replication stressor hydroxyurea. This sensitivity or toxicity is dependent on the homologous recombination (HR) system and apparently results from formation of dead-end HR DNA intermediates. HU toxicity can be suppressed by deletion of the gene encoding Brh2, the BRCA2 orthologue that serves to regulate HR by mediating Rad51 filament formation on single-stranded DNA. Brh2 harbours two different DNA-binding domains that contribute to HR function. DNA-binding activity from a single domain is sufficient to provide Brh2 functional activity in HR, but to enable HU-induced killing two functional DNA-binding domains must be present. Despite this stringent requirement for dual functioning domains, the source of DNA-binding domains is less critical in that heterologous domains can substitute for the native endogenous ones. The results suggest a model in which the nature of the DNA lesion is an important determinant in the functional response of Brh2 action.     

DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis

Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity.     

Dss1 interaction with Brh2 as a regulatory mechanism for recombinational repair

Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer-sized complexes, which in turn, can be dissociated by Dss1 to monomer. We propose that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2's molecular mechanism.     

BRCA2 homolog required for proficiency in DNA repair,recombination, and genome stability in Ustilago maydis

In a screen for DNA repair-defective mutants in the fungus Ustilago maydis, a gene encoding a BRCA2 family member, designated here as Brh2, was identified. A brh2 null allele was found to be defective in allelic recombination, meiosis, and repair of gaps and ionizing radiation damage to the same extent as rad51. Frequent marker loss in meiosis and diploid formation suggested that genomic instability was associated with brh2. This notion was confirmed by molecular karyotype analysis, which revealed gross chromosomal alterations associated with brh2. Yeast two-hybrid analysis indicated interaction between Brh2 and Rad51. Recapitulation in U. maydis of defects in DNA repair and genome stability associated with brh2 means that the BRCA2 gene family is more widespread than previously thought.     

Compensatory role for Rad52 during recombinational repair in Ustilago maydis

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.     

Inhibition of G1 to S cell cycle progression by BCCIP beta

The BCCIP alpha protein was identified as a BRCA2 and CDKN1A (p21, or p21(Waf1/Cip1)) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIP alpha enhances the inhibitory activity of p21 toward CDK2 and that BCCIP alpha inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIP beta, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIP beta can be partially abrogated in p21 deficient cells. Overexpression of BCCIP beta delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIP beta as a new regulator for the G1-S cell cycle progression and cell growth control.     

BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.     

DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells

BRCA2 is a breast cancer susceptibility gene implicated in the repair of double-strand breaks by homologous recombination with RAD51. BRCA2 associates with a 70-amino-acid protein, DSS1, but the functional significance of this interaction has remained unclear. Recently, deficiency of a DSS1 orthologue in the fungus Ustilago maydis has been shown to cause a defect in recombinational DNA repair. Here we have investigated the consequences of DSS1 depletion in mammalian cells. We show that like BRCA2, DSS1 is required for DNA damage-induced RAD51 focus formation and for the maintenance of genomic stability, indicating a function conserved from lower eukaryotes to humans. However, DSS1 seems to be not required for BRCA2 or RAD51 stability or for BRCA2 and RAD51 to interact, raising the possibility that DSS1 may be required for the BRCA2-RAD51 complex to become associated with sites of DNA damage.     

Dual DNA-binding domains shape the interaction of Brh2 with DNA

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, harbors two different DNA-binding domains, one located in the N-terminal region and the other located in the C-terminal region. Here we were interested in comparing the biochemical properties of Brh2 fragments, Brh2^NT and Brh2^CT, respectively, harboring the two different DNA-binding regions to understand the mechanistic purpose of dual DNA-interaction domains. With oligonucleotide substrates to model different DNA conformations, it was found that the substrate specificity of Brh2^NT and Brh2^CT was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2^NT. Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2^NT. Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2^NT suggesting Brh2^CT contributes to the activity when RPA is present. Brh2^NT and Brh2^CT were both found capable of physically interacting with RPA. The results suggest that while the two DNA-binding regions of Brh2 appear functionally redundant in certain aspects of DNA repair, they differ in fundamental properties, and likely contribute in different ways to repair processes involving or arising from stalled DNA replication forks.     

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.     

The beta-isoform of the BRCA2 and CDKN1A(p21)-interacting protein (BCCIP) stabilizes nuclear RPL23/uL14

BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP with distinct C-terminal domains exist in humans. We show that mammalian BCCIPβ, but not BCCIPα, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans-acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPβ. Depletion of BCCIPβ reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overexpression of BCCIPβ leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPβ functions as nuclear chaperone for RPL23.     

Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.     

Poly(ADP-ribose) Polymerase (PARP-1) in Homologous Recombination and as a Target for Cancer Therapy

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumours. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumours.