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Cell biology and genetic studies have demonstrated that DNA double-strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA-templated DSB repair pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo , we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway., Competing Interests: Conflict of Interest “The authors declare that they have no conflicts of interest with the contents of this article.”

BRC is a short evolutionarily conserved sequence motif generally arranged in multiple tandem repeats that is present as a defining feature in members of the BRCA2 tumor suppressor protein family. From crystallographic studies of a co-complex, the human BRC4 was found to form a structural element that interacts with RAD51, a key component in the DNA repair machinery directed by homologous recombination. The BRC is distinguished by two tetrameric sequence modules with characteristic hydrophobic residues separated by an intervening spacer region marked by certain highly conserved residues forming a hydrophobic surface for interaction with RAD51. It is present as a single copy in Brh2 of Ustilago maydis, the only reported example of a fungal BRCA2 ortholog. By comparative sequence analysis, examples of BRCA2 orthologs were identified in other fungal phyla, some of which featured multiple tandem repeats like those found in mammals. An expeditious biological assay system was developed for evaluating the two-tetramer module model and assessing the importance of particular conserved amino acid residues of BRC contributing to Brh2 functionality in DNA repair. This work was aided by the finding that the human BRC4 repeat could substitute completely for the endogenous BRC element in Brh2, while the human BRC5 repeat could not. In a survey of point mutations of certain residues, certain BRC mutant variants termed antimorphs were identified that caused a DNA repair phenotype more severe than the null., Competing Interests: Declaration of Competing Interest The authors confirm that there is no conflict of interest, financial, or otherwise in this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. UmPot1 binds directly to Rad51 and regulates the latter's strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection., Competing Interests: The authors have declared that no competing interests exist.

Duplex telomere binding proteins exhibit considerable structural and functional diversity in fungi. Herein we interrogate the activities and functions of two Myb-containing, duplex telomere repeat-binding factors in Ustilago maydis, a basidiomycete that is evolutionarily distant from the standard fungi. These two telomere-binding proteins, UmTay1 and UmTrf2, despite having distinct domain structures, exhibit comparable affinities and sequence specificity for the canonical telomere repeats. UmTay1 specializes in promoting telomere replication and an ALT-like pathway, most likely by modulating the helicase activity of Blm. UmTrf2, in contrast, is critical for telomere protection; transcriptional repression of Umtrf2 leads to severe growth defects and profound telomere aberrations. Comparative analysis of UmTay1 homologs in different phyla reveals broad functional diversity for this protein family and provides a case study for how DNA-binding proteins can acquire and lose functions at various chromosomal locations. Our findings also point to stimulatory effect of telomere protein on ALT in Ustilago maydis that may be conserved in other systems.

Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer-protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51 D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51 WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51 D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA-protein cross-links (DPCs) are frequently occurring lesions that provoke continual threats to the integrity of the genome by interference with replication and transcription. Reactive aldehydes generated from endogenous metabolic processes or produced in the environment are sources that trigger cross-linking of DNA with associated proteins. DNA repair pathways in place for removing DPCs, or for bypassing them to enable completion of replication, include homologous recombination (HR) and replication fork remodeling (FR) systems. Here, we surveyed a set of mutants defective in known HR and FR components to determine their contribution toward maintaining resistance to chronic formaldehyde (FA) exposure in Ustilago maydis , a fungus that relies on the BRCA2-family member Brh2 as the principal Rad51 mediator in repair of DNA strand breaks. We found that, in addition to Brh2, Rad52 was also vital for resistance to FA. Deleting the gene for Rec8, a kleisin subunit of cohesin, eliminated the requirement for Brh2, but not Rad52, in FA resistance. The Rad51 K133R mutant variant that is able to bind DNA but unable to dissociate from it was able to support resistance to FA. These findings suggest a model for DPC repair and tolerance that features a specialized role for Rad52, enabling Rad51 to access DNA in its noncanonical capacity of replication fork protection rather than DNA strand transfer., (Copyright © 2018 by the Genetics Society of America.)

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1 and Mus81. We suggest these factors comprise a subpathway for channeling repair when Brh2 is compromised in its interplay with DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination., (© 2017 John Wiley & Sons Ltd.)

Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, its mediator BRCA2, and Dss1, a key regulator of BRCA2. We seek to understand the role of BRCA2 in governing the activity of Rad51 and to learn how BRCA2 function is regulated by Dss1. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical and molecular genetic manipulations and analysis. The powerful attributes of this system open the way for gaining insight into BRCA2's molecular mechanism through avenues not immediately approachable in the vertebrate systems. Here we provide protocols for preparing Brh2, Dss1, and Rad51 as reagents for use in biochemical assays to monitor function and present methods for transposon-based mutational analysis of Brh2 for use in genetic dissection of function., (© 2018 Elsevier Inc. All rights reserved.)

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, mediates delivery of Rad51 to DNA during the course of homology-directed DNA repair. Rad51 interacts with Brh2 through the highly conserved BRC element and through a second region termed CRE located at the extreme carboxy terminus. Dss1, a small intrinsically unstructured protein that interacts with Brh2, is crucial for its activity in DNA repair, but the mechanism of this regulation is uncertain. In previous studies, we found that interaction of Brh2 with DNA was strongly modulated by association with Dss1. Here we report that CRE influences interaction of Dss1 with Brh2 and that Dss1 status markedly alters interaction of Brh2 with Rad51. While it appears that a single Rad51 protomer associates with Brh2 in complex with Dss1, loss of Dss1 is accompanied by a large increase in the number of Rad51 protomers that can associate with Brh2. Concomitant with this buildup of Rad51, Brh2 loses its ability to bind DNA. These observations suggest a feedback circuit in which release of Dss1 from Brh2 as it binds DNA triggers nucleation of a short Rad51 oligomer on Brh2, which in turn promotes dissociation of Brh2 from the DNA.

A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and "trapped" the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Ku heterodimer serves in the initial step in repairing DNA double-strand breaks by the non-homologous end-joining pathway. Besides this key function, Ku also plays a role in other cellular processes including telomere maintenance. Inactivation of Ku can lead to DNA repair defects and telomere aberrations. In model organisms where Ku has been studied, inactivation can lead to DNA repair defects and telomere aberrations. In general Ku deficient mutants are viable, but a notable exception to this is human where Ku has been found to be essential. Here we report that similar to the situation in human Ku is required for cell proliferation in the fungus Ustilago maydis. Using conditional strains for Ku expression, we found that cells arrest permanently in G2 phase when Ku expression is turned off. Arrest results from cell cycle checkpoint activation due to persistent signaling via the DNA damage response (DDR). Our results point to the telomeres as the most likely source of the DNA damage signal. Inactivation of the DDR makes the Ku complex dispensable for proliferation in this organism. Our findings suggest that in U. maydis, unprotected telomeres arising from Ku depletion are the source of the signal that activates the DDR leading to cell cycle arrest., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2's amino-terminal and carboxy-terminal domains with DNA.

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., (© 2011 Blackwell Publishing Ltd.)

BRCA2 is the product of a breast cancer susceptibility gene in humans and the founding member of an emerging family of proteins present throughout the eukaryotic domain that serve in homologous recombination. The function of BRCA2 in recombination is to control RAD51, a protein that catalyzes homologous pairing and DNA strand exchange. By physically interacting with both RAD51 and single-stranded DNA, BRCA2 mediates delivery of RAD51 preferentially to sites of single-stranded DNA (ssDNA) exposed as a result of DNA damage or replication problems. Through its action, BRCA2 helps restore and maintain integrity of the genome. This review highlights recent studies on BRCA2 and its orthologs that have begun to illuminate the molecular mechanisms by which these proteins control homologous recombination.

In the phytopathogenic fungus Ustilago maydis, the dikaryotic state dominates the period of growth occurring during the infectious phase. Dikaryons are cells in which two nuclei, one from each parent cell, share a single cytoplasm for a period of time without undergoing nuclear fusion. In fungal cells, maintenance of the dikaryotic state requires an intricate cell division process that often involves the formation of a structure known as the clamp connection as well as the sorting of one of the nuclei to this structure to ensure that each daughter dikaryon inherits a balance of each parental genome. Here, we describe an atypical role of the DNA damage checkpoint kinases Chk1 and Atr1 during pathogenic growth of U. maydis. We found that Chk1 and Atr1 collaborate to control cell cycle arrest during the induction of the virulence program in U. maydis and that Chk1 and Atr1 work together to control the dikaryon formation. These findings uncover a link between a widely conserved signaling cascade and the virulence program in a phytopathogen. We propose a model in which adjustment of the cell cycle by the Atr1-Chk1 axis controls fidelity in dikaryon formation. Therefore, Chk1 and Atr1 emerge as critical cell type regulators in addition to their roles in the DNA damage response.

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., (© 2010 Blackwell Publishing Ltd.)

Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.

Accumulating evidence for Rad51-catalyzed DNA strand invasion during double-strand break repair features a 3' single-stranded tail as the preferred substrate for reaction, but paradoxically, the preferred substrate in model reactions in vitro is the 5' end. Here, we examined the Rad51-promoted 5' end invasion reaction in the presence of Brh2, the BRCA2 family protein in Ustilago maydis. Using plasmid DNA and a homologous duplex oligonucleotide with 5' protruding single-stranded tail as substrates, we found that Brh2 can stimulate Rad51 to promote the formation of a four-stranded complement-stabilized D loop. In this structure, the incoming recessed complementary strand of the oligonucleotide has switched partners and can now prime DNA synthesis using the recipient plasmid DNA as template, circumventing a lesion that blocks elongation when the 5' protruding tail serves as template for fill-in synthesis. We propose that template switching promoted by Brh2 provides a mechanism for recombination-mediated bypass of lesions blocking synthesis during DNA replication.

Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yeast, or Rqh1 in fission yeast leads to inappropriate recombination, chromosome abnormalities, and disturbed replication fork progression. Studies with yeasts have demonstrated that auxiliary gene functions can contribute in overlapping ways with Sgs1 or Rqh1 to circumvent or overcome lesions in DNA caused by certain genotoxic agents. In the combined absence of these functions, recombination-mediated processes lead to severe loss of fitness. Here we performed a genetic study to determine the role of the Ustilago maydis Blm homolog in DNA repair and in alleviating replication stress. We characterized the single mutant as well as double mutants additionally deleted of genes encoding Srs2, Fbh1, Mus81, or Exo1. Unlike yeasts, neither the blm srs2, blm exo1, nor blm mus81 double mutant exhibited extreme loss of fitness. Inactivation of Brh2, the BRCA2 homolog, suppressed toxicity to hydroxyurea caused by loss of Blm function. However, differential suppression by Brh2 derivatives lacking the canonical DNA-binding region suggests that the particular domain structure comprising this DNA-binding region may be instrumental in promoting the observed hydroxyurea toxicity.

The C-terminal region of Brh2 (Brh2(CT)), the BRCA2 homolog in Ustilago maydis, is highly conserved and aligns with the DSS1/DNA-binding domain (DBD) of mammalian BRCA2, while the N-terminal region (Brh2(NT)) is poorly conserved and has no obvious functional domain except for the single Rad51-interacting BRC element. Paradoxically, Brh2(NT), but not Brh2(CT), complements the DNA repair and recombination deficiency of the brh2 mutant. We show here that Brh2(NT) exhibits an unexpected DNA binding activity with properties similar to that of the full-length protein. Deletion mapping localized the region responsible for the DNA binding activity to a stretch of residues between the BRC element and the canonical DBD. A heterologous DNA-binding domain from the large subunit of replication protein A substituted for the endogenous binding region within Brh2(NT) in supporting DNA repair. Rad51-promoted strand invasion was stimulated by Brh2(NT), but required the presence of the BRC element. The findings suggest a model in which Brh2(NT) serves as the principal site for association with DNA, while the Brh2(CT) provides a means for regulation.

Brh2 plays a central role in the homologous recombination system of Ustilago maydis, mediating delivery of Rad51 to single-stranded DNA. Here we report that Brh2 can pair the displaced strand of a D loop with a complementary single-stranded DNA to form a duplexed, or double, D loop. The reaction emulates the second-end capture step envisioned in models of DNA double-strand break repair. This second-end capture reaction promoted by Brh2 proceeds efficiently when performed in the presence of Rad51 under conditions that block annealing by Rad52, or when the second single-stranded DNA substrate is replaced by double-stranded DNA. In a coupled reaction that requires extension of the D loop more than 200 nt by DNA synthesis in order to reveal a complementary region, Brh2 was also able to promote second-end capture and thus model a synthesis-dependent strand-annealing mechanism.

The Holliday junction (HJ) is a central intermediate in homologous recombination. Ip et al. (2008), in a recent issue of Nature, have identified a new subclass of the Rad2/XPG family of nucleases with the hallmark of symmetrical cleavage of HJ substrates to produce nicked duplex products.

Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

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.

Brh2, the ortholog of the BRCA2 tumor suppressor in Ustilago maydis, works hand in hand with Rad51 to promote repair of DNA by homologous recombination. Previous studies established that Brh2 can stimulate DNA strand exchange by enabling Rad51 nucleoprotein filament formation on replication protein A-coated ssDNA. But, more recently, it was noted that Brh2 has an inherent DNA annealing activity, raising the notion that it might have roles in recombination in addition to or beyond the mediator function. Here, we found that Brh2 can autonomously promote the formation of D-loops in reactions with plasmid DNA and homologous single-stranded oligonucleotides. The reaction differs from that catalyzed by Rad51 in having no requirement for cofactors or preloading phase on ssDNA. D-loop formation was most effective when Brh2 was mixed with plasmid DNA before addition of single-stranded oligomer. D-loop formation catalyzed by Rad51 was also enhanced when Brh2 was premixed with plasmid DNA. Brh2 rendered defective in Rad51 interaction by mutation in the BRC element was still capable of promoting D-loop formation. However, the mutant protein was unable to enhance the Rad51-catalyzed reaction. The results suggest a model in which Brh2 binding to plasmid DNA attracts and helps capture Rad51-coated ssDNA.

Ustilago maydis is a phytopathogenic fungus exhibiting extreme resistance to UV and ionizing radiation. The molecular mechanisms underlying this resistance are as yet unknown. The recently determined genome sequence was examined for clues to the radiation resistance, focusing on proteins in homologous recombination, but there was little that was unusual about them. Furthermore, by comparison, its recombinational repair system seems to be only minimally related to the extended synthesis-dependent DNA strand-annealing system of Deinococcus radiodurans. Thus, consideration should be given to the possibility that incremental structural changes in repair proteins or their elevated expression are the basis for the extreme radiation resistance in U. maydis. Evolution of a system enabling the survival of U. maydis under such conditions could be a secondary consequence of adaptation to an environment of continual genotoxic stress encountered in its habitat.

The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulating the action of Rad51. In turn, BRCA2 appears to be regulated by other interacting proteins. Dss1, a small interacting protein that binds to the C-terminal domain, has a profound effect on activity as deduced from studies on the BRCA2-related protein Brh2 in Ustilago maydis. Evidence accumulating in mammalian systems suggests that BCCIP, another small interacting protein that binds to the C-terminal domain of BRCA2, also serves to regulate homologous recombination activity. Here we were interested in testing the role of the putative U. maydis BCCIP ortholog Bcp1 in DNA repair and recombination. In keeping with the mammalian paradigm, Bcp1 bound to the C-terminal region of Brh2. Mutants deleted of the gene were extremely slow growing, showed a delay passing through S phase and exhibited sensitivity to hydroxyurea, but were otherwise normal in DNA repair and homologous recombination. In the absence of Bcp1 cells were unable to maintain the wild type morphology when challenged by a DNA replication stress. These results suggest that Bcp1 could be involved in coordinating morphogenetic events with DNA processing during replication.

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.

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.

Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.

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.

The BRCA2 tumour suppressor is essential for the error-free repair of double-strand breaks (DSBs) in DNA by homologous recombination. This is mediated by RAD51, which forms a nucleoprotein filament with the 3' overhanging single-stranded DNA (ssDNA) of the resected DSB, searches for a homologous donor sequence, and catalyses strand exchange with the donor DNA. The 3,418-amino-acid BRCA2 contains eight approximately 30-amino-acid BRC repeats that bind RAD51 (refs 5, 6) and a approximately 700-amino-acid DBD domain that binds ssDNA. The isolated BRC and DBD domains have the opposing effects of inhibiting and stimulating recombination, respectively, and the role of BRCA2 in repair has been unclear. Here we show that a full-length BRCA2 homologue (Brh2) stimulates Rad51-mediated recombination at substoichiometric concentrations relative to Rad51. Brh2 recruits Rad51 to DNA and facilitates the nucleation of the filament, which is then elongated by the pool of free Rad51. Brh2 acts preferentially at a junction between double-stranded DNA (dsDNA) and ssDNA, with strict specificity for the 3' overhang polarity of a resected DSB. These results establish a BRCA2 function in RAD51-mediated DSB repair and explain the loss of this repair capacity in BRCA2-associated cancers.

The tumor suppressor BRCA2 plays an essential role in the repair of double-strand DNA breaks by regulating the action of the RAD51 recombinase. The activity of BRCA2 is in turn governed by DSS1, a small acidic protein that appears to function as a necessary cofactor. A model fungal system that reproduces the BRCA2-RAD51 interaction offers the opportunity to understand the mechanism of DSS1 activation at the molecular level.

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.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes appears to involve the generation of double-strand DNA breaks (DSBs) and their error-prone repair. Here we show that DSBs occur at a high frequency in unrearranged (germline) Ig variable (V) genes, BCL6 and c-MYC. These DSBs are blunt, target the mutational RGYW/RGY hotspot, and would be resolved through nonhomologous end-joining, as indicated by the presence of Ku70/Ku86 on these DNA ends. Upon CD40-induced expression of activation-induced cytidine deaminase (AID), DSBs increase in frequency and are resected to yield 5'- and 3'-protruding ends in hypermutating rearranged V genes, BCL6 and translocated c-MYC. 3'-protruding ends would direct DSB repair through homologous recombination, as indicated by their exclusive presence in S/G2 and recruitment of Rad52/Rad51, leading to SHM, upon mispair by error-prone DNA polymerases modulated by crosslinking of the B cell receptor for antigen.

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.