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Abstract: Base excision repair (BER) of dimethyl sulfate induced N-methylpurines (NMPs) was measured at nucleotide resolution in the mitochondrial DNA (mtDNA) of cultured human and yeast (Saccharomyces cerevisiae) cells. NMPs were repaired with heterogeneous rates in the human mtDNA. The nearest-neighbor nucleotides significantly affected the repair rates: NMPs between pyrimidines were repaired much faster than those between purines, and those between a purine and a pyrimidine were repaired at intermediate rates. Repair intermediates of NMPs can also be detected at certain sites of the human mtDNA, indicating an ineffectiveness of processing the intermediates at these sites by the human mitochondrial BER machinery. In contrast to the human mtDNA, the yeast mtDNA did not show detectable repair of NMPs at any sites. Furthermore, a high level of spontaneous strand breaks exists exclusively at purine sites in the yeast mtDNA. Spontaneous NMPs or oxidative lesions were unlikely to be the major causes for the spontaneous strand breaks. Rather, spontaneous depurination combined with inefficient processing of DNA nicks or single-nucleotide gaps by the yeast mitochondrial BER machinery may result in the spontaneous strand breaks. Our results unveil a striking difference in BER between human and the yeast mitochondria. [ABSTRACT FROM AUTHOR]

Abstract: Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. Rad26, a member of the Swi2/Snf2 superfamily of proteins, has been shown to be involved in a specialized NER process called transcription coupled NER. Rad16, another member of the same protein superfamily, has been shown to be required for genome-wide NER. Here we show that Rad16 and Rad26 play different roles in repairing repressed and actively transcribed genes in yeast. Rad16 is partially dispensable, and Rad26 plays a significant role in repairing certain regions of the repressed GAL1-10, PHO5 and ADH2 genes, especially in the core DNA of well-positioned nucleosomes. Simultaneous elimination of Rad16 and Rad26 results in no detectable repair in these regions of the repressed genes. Transcriptional induction of the GAL1-10 genes abolishes the role of Rad26, but does not affect the role of Rad16 in repairing the nontranscribed strand of the genes. Interestingly, when the transcription activator Gal4 is eliminated from the cells, Rad16 becomes partially dispensable and Rad26 plays a significant role in repairing both strands of the GAL1-10 genes even under inducing conditions. Our results suggest that Rad16 and Rad26 play different and, to some extent, complementary roles in repairing both strands of repressed genes, although the relative contributions of the two proteins can be different from gene to gene, and from region to region of a gene. However, Rad16 is solely responsible for repairing the nontranscribed strand of actively transcribed genes. [Copyright &y& Elsevier]

Abstract: Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of an actively transcribed gene. To be distinguished from TCR-NER, the genome-wide NER process is termed as global genomic NER (GG-NER). In Saccharomyces cerevisiae, GG-NER is dependent on Rad7, whereas TC-NER is mediated by Rad26, the homolog of the human Cockayne syndrome group B protein, and by Rpb9, a non-essential subunit of RNA polymerase II. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy. Here, we show that Tfb5 plays different roles in different NER pathways in yeast. No repair takes place in the non-transcribed strand of a gene in tfb5 cells, or in both strands of a gene in rad26 rpb9 tfb5 cells, indicating that Tfb5 is essential for GG-NER. However, residual repair occurs in the transcribed strand of a gene in tfb5 cells, suggesting that Tfb5 is important, but not absolutely required for TC-NER. Interestingly, substantial repair occurs in the transcribed strand of a gene in rad7 tfb5 and rad7 rpb9 tfb5 cells, indicating that, in the absence of GG-NER, Tfb5 is largely dispensable for Rad26 mediated TC-NER. Furthermore, we show that no repair takes place in the transcribed strand of a gene in rad7 rad26 tfb5 cells, suggesting that Tfb5 is required for Rpb9 mediated TC-NER. Taken together, our results indicate that Tfb5 is partially dispensable for Rad26 mediated TC-NER, especially in GG-NER deficient cells. However, this TFIIH subunit is required for other NER pathways. [Copyright &y& Elsevier]