Freyer G A, Davey S, Ferrer J V, Martin A M, Beach D, Doetsch P W
Center for Radiological Research, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Mol Cell Biol. 1995 Aug;15(8):4572-7. doi: 10.1128/MCB.15.8.4572.
DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.
紫外线、环丁烷嘧啶二聚体和（6-4）嘧啶嘧啶酮诱导产生的DNA损伤可通过核苷酸切除修复（NER）过程进行修复。然而，在裂殖酵母粟酒裂殖酵母中，研究表明至少存在两种切除紫外线光产物的机制；NER和另一种以前未被识别的过程。最近我们报道，粟酒裂殖酵母含有一种DNA内切酶SPDE，它能识别并在紧邻环丁烷嘧啶二聚体和（6-4）嘧啶嘧啶酮的位置进行切割。在此我们报道，对紫外线敏感的粟酒裂殖酵母rad12-502突变体缺乏SPDE活性。此外，如体外切除修复试验所示，从rad12-502突变体制备的提取物在DNA切除修复方面存在缺陷。通过向体外修复反应混合物中添加部分纯化的SPDE，从rad12-502细胞制备的提取物中的DNA修复活性恢复到了野生型水平。当rad12-502突变体与NER rad13-A突变体杂交时，产生的双突变体对紫外线辐射的敏感性比任何一个单突变体都要高得多，这表明rad12基因产物在一条与NER不同的DNA修复途径中发挥作用。这些数据直接将SPDE与这种替代切除修复过程联系起来。我们提出，依赖SPDE的DNA修复途径是粟酒裂殖酵母中存在的第二种DNA切除修复过程。
