Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of doublestrand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly-efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300 bp and 60 bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.
To precisely define the functional sequence of the CHOI gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the originai 4.5-kilobase (kb) CHOI clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of chol mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol, led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression., Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHOI gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opil, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When chol mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in chol cells.
A 34 base-pair (bp) fragment spanning sequences -154 to -120 of the promoter of the CHO1 gene (structural gene for phosphatidylserine synthase) from the yeast Saccharomyces cerevisiae has been shown to place transcription of a promoter-less Escherichia coli lacZ gene under control of the phospholipid precursors inositol and choline. Furthermore, in deletion experiments the CHO1 UASINO was localized to sequences between -151 and -123 of the CHO1 promoter. A nine bp sequence was identified in the promoter region of the CHO1 gene that shares an eight out of nine bp match with a sequence (consensus 5' ATGTGAAAT 3') that is repeated a total of 23 times upstream from several coregulated phospholipid biosynthetic genes. This sequence is contained within the -151 to -123 region to which the CHO1 UAS has been localized. The nine bp repeated element is believed to be involved in the control of phospholipid biosynthetic gene transcription in response to changing levels of inositol and choline in the growth medium. This control has been shown to require activities encoded by the products of the three regulatory genes: INO2, INO4, and OPI1. A mutation in any of these regulatory genes results in aberrant CHO1-lacZ gene regulation, and affects regulation of the construct containing the 34 bp (-154 to -120) CHO1 fragment demonstrating that the regulatory signal generated by these genes interacts with the 5' end of the CHO1 gene.
Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.
BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5′ UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.
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