Abstract
The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.
Highlights
Rad3, the Saccharomyces cerevisiae homolog of human XPD, is a 5’ to 3’ DNA helicase that is a subunit of the TFIIH RNA polymerase II initiation factor complex [1,2]
The two members of each pair of homologous chromosomes in diploid eukaryotes are usually heterozygous for many single-nucleotide polymorphisms (SNPs)
Mitotic recombination can be beneficial as a source of genetic diversity, loss of wild-type alleles of tumor suppressor genes in heterozygous individuals is an important contributor to carcinogenesis
Summary
The Saccharomyces cerevisiae homolog of human XPD, is a 5’ to 3’ DNA helicase that is a subunit of the TFIIH RNA polymerase II initiation factor complex [1,2]. As part of the TFIIH complex, it has roles in transcription initiation and nucleotide excision repair (NER) [3]. During NER, Rad3/XPD unwinds the DNA containing a UV-induced lesion, producing double-stranded to single-stranded transitions that are substrates for the endnucleases Rad1-10/XPF-ERCC1 and Rad2/XPG [6]. These nucleases make cuts flanking the lesion, allowing its removal. The resulting single-stranded gap is filled by DNA polymerase, followed by ligation to complete the repair
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