Suppressors of Blm-deficiency identify three novel proteins that facilitate DNA repair in Ustilago maydis.

To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm-KR hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the "normally" growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-KR, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1.

Identification of Genes Promoting Growth of Ustilago maydis on Biomolecules Released from Cells Killed by Oxidation.

Much headway has been made in understanding the numerous strategies that enable microorganisms to counteract various types of environmental stress, but little is known about how microbial populations recover after a massive death caused by exposure to extreme conditions. Using the yeast-like fungus Ustilago maydis as a model, our recent post-stress regrowth under starvation (RUS) studies have demonstrated that this organism reconstitutes devastated populations with remarkable efficiency. Subsequently, we have identified four RUS-gene products. Two of these, Did4 and Tbp1, play parallel roles in protecting the genome. To identify additional molecular components, we took a molecular-genetic and a transcriptomic approach. By employing a simple and novel screening method, we identified five RUS-deficient mutants (snf8, slm1, vrg4, snf5, hsf1), three of which (snf8, slm1, and hsf1) displayed sensitivity to different genotoxic agents, indicating that the corresponding gene products have roles in genome protection. The global transcriptomic changes of cells grown in supernatants derived from peroxide-treated cell suspensions revealed sets of uniquely expressed genes. Importantly, among the genes induced by the substrates was Chk1, which encodes a protein kinase required for checkpoint-mediated cell cycle arrest in response to DNA damage. Mutants of U. maydis deleted of Chk1 are severely incapacitated in RUS.

Self-Generated Hypoxia Leads to Oxidative Stress and Massive Death in Ustilago maydis Populations under Extreme Starvation and Oxygen-Limited Conditions.

Ustilago maydis and Saccharomyces cerevisiae differ considerably in their response to water-transfer treatments. When stationary phase cells were transferred to pure water and incubated under limited supply of oxygen, the U. maydis cells suffered a catastrophic loss of viability while the S. cerevisiae population was virtually unaffected by the treatment. The major factor underlying the death of the U. maydis cells under those conditions was an oxygen-consuming cellular activity that generated a hypoxic environment, thereby inducing oxidative stress and accumulation of reactive oxygen species, which resulted in lethality. Importantly, a small residue of U. maydis cells that did survive was able to resume growth and repopulate up to the initial culture density when sufficient aeration was restored. The regrowth was dependent on the cellular factors (Adr1, Did4, Kel1, and Tbp1), previously identified as required for repopulation, after killing with hydrogen peroxide. Surprisingly, the survivors were also able to resume growth under apparently hypoxic conditions, indicating that these remnant cells likely switched to a fermentative mode of growth. We discuss the findings in terms of their possible relevance to the eco-evolutionary adaptation of U. maydis to risky environments.

When disaster strikes: Reconstitution of population density by expansion of survivors.

Microorganisms have an assortment of stress-response mechanisms that enable them to survive in the face of environmental stresses. However, with prolonged exposures to severe stresses adaptive stress responses ultimately fail, the affected populations may suffer a massive decline. Recovery of the population density in the aftermath of a massive death is a vital task. Our recent post-stress regrowth under starvation (RUS) studies prompted us to propose RUS as an adaptation for overcoming consequences of devastating environmental disturbances. RUS should be seen as an integral process having two major aspects: the stress-induced cellular auto-decomposition and the recycling of the released nutrients. Here, we summarized what is already known about RUS and suggest a number of questions that are key to understanding the molecular underpinnings of these two operations. We also interrogate the prospect that would conceptualize the auto-decomposition as a fitness-maximizing mechanism acting with the purpose of an expedient supply of nutrients. Two further things are of special note: given that some of the RUS-defective mutants are also impaired in DNA repair, RUS can serve as an important tool for uncovering new determinants operating, in some overlapping fashion, in the protection of genome integrity; also, RUS can serve as a new angle of approach that might, hopefully, assign roles to some of those (up to ~ 30%) of microbial genes that are of unknown function. More generally, understanding post-stress reconstitution and the underlying mechanisms is a necessary (complementing) part of any comprehensive picture of how microbes cope with very harsh environmental disturbances.

A comparative study of liquid holding restitution of viability after oxidative stress in Ustilago maydis and Saccharomyces cerevisiae cell populations.

The ability of Saccharomyces cerevisiae to reconstitute viability after strong peroxide-induced oxidative stress during liquid holding (LH) in non-nutrient medium has been compared with that of Ustilago maydis. It was found that like U. maydis, S. cerevisiae was capable of reconstituting viability through multiplication of the survivors. However, differences were observed in the pattern of their response: (i) the reconstitution of viability was slower in S. cerevisiae; (ii) before the viability was progressively increasing the treated samples of this fungus reproducibly passed through a phase of additional decrease of the surviving fraction and (iii) the final yields of viable cells attained in S. cerevisiae were below those achieved by U. maydis. The reason for the relative superiority of U. maydis is twofold: (1) early initiated and faster degradation and leakage of the intracellular biomolecules and (2) greater ability of U. maydis cells to recycle damaged and released intracellular compounds. Conceptually similar studies extended to another oxidative-stress-inducing condition, namely desiccation, indicated that the marked differences between these fungi in their patterns of the post-stress regrowth, cellular leakage and macromolecule decomposition are reproduced during LH of desiccated cells. The concordance of the findings obtained upon these two approaches was also corroborated by an analysis of the post-desiccation LH response of U. maydis mutants (adr1, did4, kel1 and tbp1) that were previously identified as defective in post-peroxide LH restitution of viability. We discuss the findings in terms of their possible relevance to the mechanisms of the ecological and evolutionary adaptation of free-living microorganisms to fluctuating and severely inhospitable environments.

Bioavailability of Nutritional Resources From Cells Killed by Oxidation Supports Expansion of Survivors in Ustilago maydis Populations.

After heavy exposure of Ustilago maydis cells to clastogens, a great increase in viability was observed if the treated cells were kept under starvation conditions. This restitution of viability is based on cell multiplication at the expense of the intracellular compounds freed from the damaged cells. Analysis of the effect of the leaked material on the growth of undamaged cells revealed opposing biological activity, indicating that U. maydis must possess cellular mechanisms involved not only in reabsorption of the released compounds from external environment but also in contending with their treatment-induced toxicity. From a screen for mutants defective in the restitution of viability, we identified four genes (adr1, did4, kel1, and tbp1) that contribute to the process. The mutants in did4, kel1, and tbp1 exhibited sensitivity to different genotoxic agents implying that the gene products are in some overlapping fashion involved in the protection of genome integrity. The genetic determinants identified by our analysis have already been known to play roles in growth regulation, protein turnover, cytoskeleton structure, and transcription. We discuss ecological and evolutionary implications of these results.

Approaches to Understanding the Mediator Function of Brh2 in Ustilago maydis.

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.

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 subpathway for channeling repair when Brh2 is compromised in its interplay with DNA.

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 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.

The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

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 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.

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.

Dss1 release activates DNA binding potential in Brh2.

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.

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.

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.

Dss1 regulates interaction of Brh2 with DNA.

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.

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.

DNA-binding Domain within the Brh2 N Terminus Is the Primary Interaction Site for Association with DNA.

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.

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.

Ortholog of BRCA2-interacting protein BCCIP controls morphogenetic responses during DNA replication stress in Ustilago maydis.

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.