The sisomicin-gentamicin resistance methylase {sgm) gene from Micromonospora zionensis (producer ... more The sisomicin-gentamicin resistance methylase {sgm) gene from Micromonospora zionensis (producer of G-52 antibiotic), encodes an enzyme that modifies 16S rRNA, and thereby conferring resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Two promoters were identified upstream of the sgm coding sequence. One promoter (P1) is relatively weak and initiates transcription at the G which is 72 nucleotides upstream of the translational initiation codon (ATG). A second (P2) promoter is much stronger and is located more upstream from the ATG codon (-250 nucleotides). These tandem promoters could enable differential gene expression of the sgm gene, and it has been postulated that promoters have specialized functions in the producing organism. The apparent weak P1 promoter is probably responsible for constitutive transcription of the sgm gene, whereas the P2 promoter may play a specialized role in expression of the downstream (biosynthetic) genes, during the time that the antibiotic G-52 is produced. Therefore, P2 promoter could be potentially used in expressing cloned genes especially during the stationary growth phase of micromonospora. A set of transcriptional and translational fusions of the sgm gene and the lacZ reporter gene have been constructed by using the pPLtl7G plasmid. Both transcriptional and translational fusions were transcribed under the strong PLtl promoter, since it has been shown that the sgm promoters are non-functional in E. coli. Transformants harbouring either the transcriptional and translational fusions were assayed for B-galactosidase activity. Translational fusion transformants which also carried an extra copy of sgm gene either in c/s or in trans position, exhibited a substantial decrease in (3-galactosidase activity. No significant effect was observed in comparable experiments with the transcriptional fusions. These results demonstrate that the expression of the sgm gene is regulated by translational autorepression, presumably due to methylase binding to the specific site/s on its own mRNA. The translational repression model is discussed in light of overall control of the sgm gene expression. The expression of the sgm gene in various actinomycètes strains revealed that expression of high-level resistance to hygromycin B, determined by sgm gene, is background dependent. This evidence suggests that it may well be that all tested micromonospora strains share a common mode of hygromycin B resistance. Factors that might be contributed to background-dependent expression of hygromycin B resistance are discussed.
Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA interm... more 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.
Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose func... more 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.
Microorganisms have an assortment of stress-response mechanisms that enable them to survive in th... more 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.
Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by ... more 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.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The tumor suppressor BRCA2 plays an essential role in repair of double-strand DNA breaks by regul... more The tumor suppressor BRCA2 plays an essential role in repair of double-strand DNA breaks by regulating the action of the RAD51 recombinase. The activity of BRCA2 in turn is 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 at the molecular level the mechanism of DSS1 activation.
The C-terminal region of Brh2 (Brh2 CT), the BRCA2 homolog in Ustilago maydis, is highly conserve... more 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 Rad51interacting 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.
Mutants of the fungus Ustilago maydis defective in the RecQ helicase Blm are highly sensitive to ... more 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 ortholog that serves to regulate HR by mediating Rad51 filament formation on singlestranded DNA. Brh2 harbors 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.
A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago... more 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 spo11D 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.
Shuttle vectors with new or improved features were constructed to enable facile genetic manipulat... more Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.
Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yea... more 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.
A single Rad52-related protein is evident by BLAST analysis of the Ustilago maydis genome databas... more 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 crosslinking 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.
terized by missing and fused digits and by additional abnormalities affecting skin, genitourinary... more terized by missing and fused digits and by additional abnormalities affecting skin, genitourinary, and craniofacial structures (Zlotogora, 1994). An autosomal dominant form of this disorder (SHFM1) involves deletions of a minimal 5.1ف Mb locus that contains DSS1 (Crack
Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling... more 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.
The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was... more The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV‐sensitive mutants. The original isolate, rec2‐1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis. We report here that the rec2‐197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2. The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2‐197 allele that augments or interferes with REC2 functions. This product appears to be a polypeptide expressed from a remnant of the 5′ end of the open reading frame that was not removed in creating the gene disruption. The mutator activity and disturbed meiosis of rec2‐197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation. A prediction based on the phenotype of rec2‐197 is that Rec2 protein interacts with one or more other proteins in directing these functions. To identify interacting proteins we performed a yeast two‐hybrid screen and found Rad51 as a candidate. Rec2‐197 and Rad51 appear to interact to a similar degree.
The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulat... more 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 Cterminal 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.
Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radi... more 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.
1998b; Wong et al., 1997). Expression of DNA fragments encoding individual BRC repeats disrupts t... more 1998b; Wong et al., 1997). Expression of DNA fragments encoding individual BRC repeats disrupts the BRCA2-Rad51 complex, conferring a dominant-negative radiation hypersensitivity and an inability to form radiationinduced Rad51 nuclear foci (Chen et al., 1999). Synthetic
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The sisomicin-gentamicin resistance methylase {sgm) gene from Micromonospora zionensis (producer ... more The sisomicin-gentamicin resistance methylase {sgm) gene from Micromonospora zionensis (producer of G-52 antibiotic), encodes an enzyme that modifies 16S rRNA, and thereby conferring resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Two promoters were identified upstream of the sgm coding sequence. One promoter (P1) is relatively weak and initiates transcription at the G which is 72 nucleotides upstream of the translational initiation codon (ATG). A second (P2) promoter is much stronger and is located more upstream from the ATG codon (-250 nucleotides). These tandem promoters could enable differential gene expression of the sgm gene, and it has been postulated that promoters have specialized functions in the producing organism. The apparent weak P1 promoter is probably responsible for constitutive transcription of the sgm gene, whereas the P2 promoter may play a specialized role in expression of the downstream (biosynthetic) genes, during the time that the antibiotic G-52 is produced. Therefore, P2 promoter could be potentially used in expressing cloned genes especially during the stationary growth phase of micromonospora. A set of transcriptional and translational fusions of the sgm gene and the lacZ reporter gene have been constructed by using the pPLtl7G plasmid. Both transcriptional and translational fusions were transcribed under the strong PLtl promoter, since it has been shown that the sgm promoters are non-functional in E. coli. Transformants harbouring either the transcriptional and translational fusions were assayed for B-galactosidase activity. Translational fusion transformants which also carried an extra copy of sgm gene either in c/s or in trans position, exhibited a substantial decrease in (3-galactosidase activity. No significant effect was observed in comparable experiments with the transcriptional fusions. These results demonstrate that the expression of the sgm gene is regulated by translational autorepression, presumably due to methylase binding to the specific site/s on its own mRNA. The translational repression model is discussed in light of overall control of the sgm gene expression. The expression of the sgm gene in various actinomycètes strains revealed that expression of high-level resistance to hygromycin B, determined by sgm gene, is background dependent. This evidence suggests that it may well be that all tested micromonospora strains share a common mode of hygromycin B resistance. Factors that might be contributed to background-dependent expression of hygromycin B resistance are discussed.
Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA interm... more 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.
Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose func... more 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.
Microorganisms have an assortment of stress-response mechanisms that enable them to survive in th... more 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.
Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by ... more 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.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The tumor suppressor BRCA2 plays an essential role in repair of double-strand DNA breaks by regul... more The tumor suppressor BRCA2 plays an essential role in repair of double-strand DNA breaks by regulating the action of the RAD51 recombinase. The activity of BRCA2 in turn is 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 at the molecular level the mechanism of DSS1 activation.
The C-terminal region of Brh2 (Brh2 CT), the BRCA2 homolog in Ustilago maydis, is highly conserve... more 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 Rad51interacting 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.
Mutants of the fungus Ustilago maydis defective in the RecQ helicase Blm are highly sensitive to ... more 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 ortholog that serves to regulate HR by mediating Rad51 filament formation on singlestranded DNA. Brh2 harbors 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.
A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago... more 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 spo11D 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.
Shuttle vectors with new or improved features were constructed to enable facile genetic manipulat... more Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.
Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yea... more 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.
A single Rad52-related protein is evident by BLAST analysis of the Ustilago maydis genome databas... more 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 crosslinking 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.
terized by missing and fused digits and by additional abnormalities affecting skin, genitourinary... more terized by missing and fused digits and by additional abnormalities affecting skin, genitourinary, and craniofacial structures (Zlotogora, 1994). An autosomal dominant form of this disorder (SHFM1) involves deletions of a minimal 5.1ف Mb locus that contains DSS1 (Crack
Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling... more 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.
The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was... more The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV‐sensitive mutants. The original isolate, rec2‐1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis. We report here that the rec2‐197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2. The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2‐197 allele that augments or interferes with REC2 functions. This product appears to be a polypeptide expressed from a remnant of the 5′ end of the open reading frame that was not removed in creating the gene disruption. The mutator activity and disturbed meiosis of rec2‐197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation. A prediction based on the phenotype of rec2‐197 is that Rec2 protein interacts with one or more other proteins in directing these functions. To identify interacting proteins we performed a yeast two‐hybrid screen and found Rad51 as a candidate. Rec2‐197 and Rad51 appear to interact to a similar degree.
The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulat... more 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 Cterminal 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.
Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radi... more 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.
1998b; Wong et al., 1997). Expression of DNA fragments encoding individual BRC repeats disrupts t... more 1998b; Wong et al., 1997). Expression of DNA fragments encoding individual BRC repeats disrupts the BRCA2-Rad51 complex, conferring a dominant-negative radiation hypersensitivity and an inability to form radiationinduced Rad51 nuclear foci (Chen et al., 1999). Synthetic
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
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Papers by Milorad Kojic