Tung B S, McGregor W G, Wang Y C, Maher V M, McCormick J J
Carcinogenesis Laboratory, Fee Hall, Department of Microbiology, Michigan State University, East Lansing 48824-1316, USA.
Mutat Res. 1996 Jan 2;362(1):65-74. doi: 10.1016/0921-8777(95)00034-8.
Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts from such patients are extremely sensitive to mutations induced by UV radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (HPRT) gene differs significantly from that seen in normal cells. To determine if this UV hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (CPD) or 6-4 pyrimidine-pyrimidones (6-4s) in that gene, we synchronized XP variant and normal fibroblasts, irradiated them in early G1-phase, 12 or more hours prior to the scheduled onset of S phase, harvested them immediately or after allowing various times for repair, and analyzed the DNA for photoproducts in the HPRT gene, using quantitative Southern blotting. To incise the DNA at CPD, we used T4 endonuclease V; to incise at 6-4s, we first used photolyase and UV365nm to reverse CPD and then UvrABC excinuclease. Excision of CPD was rapid, preferential, and strand-specific, but there was no significant difference in rate between the two kinds of cells. The half life was 4 h in the transcribed strand of the gene and 6.5 h in the nontranscribed strand. For excision of CPD in the genome overall, this value is 12 h. Excision of 6-4s from either strand of the HPRT gene was extremely rapid and preferential in both kinds of cells, with a half life of approximately 30 min. The results indicate that the UV hypermutability of the XP variant cells cannot be caused by slower rates of repair of CPD and/or 6-4s in the target gene for mutagenesis.
着色性干皮病（XP）变异型患者在遗传上易患阳光诱发的皮肤癌。来自此类患者的成纤维细胞对紫外线辐射诱导的突变极为敏感，并且其次黄嘌呤磷酸核糖基转移酶（HPRT）基因中诱导的突变谱与正常细胞中所见的有显著差异。为了确定这种紫外线高突变性是否反映了该基因中环丁烷嘧啶二聚体（CPD）或6-4嘧啶-嘧啶酮（6-4s）的异常缓慢切除修复，我们同步了XP变异型和成纤维细胞，在预定的S期开始前12小时或更长时间的G1期早期对它们进行照射，立即或在允许进行不同时间的修复后收获它们，并使用定量Southern印迹法分析HPRT基因中的DNA光产物。为了在CPD处切割DNA，我们使用了T4内切酶V；为了在6-4s处切割，我们首先使用光解酶和UV365nm来逆转CPD，然后使用UvrABC核酸外切酶。CPD的切除是快速、优先且具有链特异性的，但两种细胞之间的切除速率没有显著差异。在基因的转录链中半衰期为4小时，在非转录链中为6.5小时。对于整个基因组中CPD的切除，这个值是12小时。从HPRT基因的任何一条链上切除6-4s在两种细胞中都极其快速且优先，半衰期约为30分钟。结果表明，XP变异型细胞的紫外线高突变性不可能是由诱变靶基因中CPD和/或6-4s的修复速率较慢引起的。
