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.
The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.
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.
RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.
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