Verhage R A, van Gool A J, de Groot N, Hoeijmakers J H, van de Putte P, Brouwer J
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Mol Cell Biol. 1996 Feb;16(2):496-502. doi: 10.1128/MCB.16.2.496.
The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.
核苷酸切除修复（NER）途径被认为由两个子途径组成：转录偶联修复，仅限于活性基因的转录链；以及针对非转录DNA链的全基因组修复。最近我们克隆了RAD26基因，它是人类CSB/ERCC6基因在酿酒酵母中的同源物，该人类基因参与转录偶联修复以及科凯恩综合征。本文描述了对在每个NER子途径中受到选择性影响的酵母双突变体的分析。虽然rad26缺失突变体在转录偶联修复方面存在缺陷，但它们对紫外线不敏感。然而，RAD26与全基因组修复决定因素RAD7和RAD16的双突变体比单个rad7或rad16突变体对紫外线更敏感，但不如完全缺乏NER的突变体敏感。这些发现揭示了RAD26和转录偶联修复在紫外线存活中的作用，表明转录偶联修复和全基因组修复部分重叠，并为双突变体中残留的NER模式提供了证据。对rad7/16 rad26双突变体中活性RPB2基因二聚体去除的分析揭示：（i）全基因组修复因子Rad7p和Rad16p对转录链修复的贡献，证实了两个NER子途径之间的部分重叠；（ii）转录链的特异性残留修复。为了研究这种修复活性对转录的依赖性，研究了可诱导的GAL7基因的链特异性修复。该基因的模板链仅在诱导条件下被修复，这表明转录在双突变体的残留修复中起作用，并表明转录偶联修复在一定程度上可以独立于Rad26p发挥作用。我们的发现还表明转录偶联修复对RAD26的依赖性存在基因座异质性。
