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.This is the peer reviewed version of the paper: Kojic, M., & Holloman, W. K. (2012). Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress. Molecular Microbiology, 83(2), 351–361.[ https://doi.org/10.1111/j.1365-2958.2011.07935.x]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/584

Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis

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 sub pathway for channeling repair when Brh2 is compromised in its interplay with DNA.Published version: [https://imagine.imgge.bg.ac.rs/ha]ndle/123456789/1190This is the peer reviewed version of the paper: Kojic, M., Milisavljevic, M., & Holloman, W. K. (2018). Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis. DNA Repair, 63, 47–55.[ https://doi.org/10.1016/j.dnarep.2018.01.010

Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis

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.This is the peer reviewed version of the paper: Mao, N., Kojić, M., & Holloman, W. K. (2009). Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis. DNA Repair, 8(6), 752–759. [https://doi.org/10.1016/j.dnarep.2009.02.006]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/343

Mutational analysis of Brh2 reveals requirements for compensating mediator functions

This is the peer-reviewed version of the article: Kojic, M., Zhou, Q., Fan, J., & Holloman, W. K. (2011). Mutational analysis of Brh2 reveals requirements for compensating mediator functions. Molecular Microbiology, 79(1), 180–191.[ https://doi.org/10.1111/j.1365-2958.2010.07440.x]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/476

Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres

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.This is the peer reviewed version of the paper: Yu, E. Y., Kojic, M., Holloman, W. K., & Lue, N. F. (2013). Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres. DNA Repair, 12(7), 472–479.[ https://doi.org/10.1016/j.dnarep.2013.04.027]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/653

Initiation of Meiotic Recombination in Ustilago maydis

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 spo11 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

Mre11 and blm-dependent formation of ALT-like telomeres in ku-deficient Ustilago maydis

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.The authors received funding from the following sources: The Meyer Cancer Center of Weill Cornell Medical College URL:http://meyercancer.weill.cornell.edu/) to NFL and WKH, The Bohmfalk Chritable Trust to NFL, and the Spanish government (BIO2014-55398-R) to JPM.Peer Reviewe

LAMMER kinase contributes to genome stability in Ustilago maydis

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 carboxyterminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1 Delta) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*) rad51 Delta 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 lkh1 Delta and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1 Delta mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1 Delta 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.This is the peer reviewed version of the paper: de Sena-Tomás, C., Sutherland, J. H., Milisavljevic, M., Nikolic, D. B., Pérez-Martín, J., Kojic, M., & Holloman, W. K. (2015). LAMMER kinase contributes to genome stability in Ustilago maydis. DNA Repair, 33, 70–77.[ https://doi.org/10.1016/j.dnarep.2015.05.011]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/887

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.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1621