Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.
Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.
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DOI：
10.1073/pnas.88.17.7810
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发表时间：
1991-09
影响因子：
11.1
通讯作者：
Y. Wang;V. Maher;J. Mccormick
中科院分区：
文献类型：
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作者：
Y. Wang;V. Maher;J. Mccormick
着色性干皮病 (XP) 变异患者表现出该疾病的临床特征，即皮肤癌发生率增加，但他们的细胞对紫外线引起的 DNA 损伤的核苷酸切除修复率正常或接近正常，并且对紫外线辐射的细胞毒性作用仅比正常细胞稍微敏感一些。然而，它们对其诱变作用明显更加敏感。为了检查造成这种超突变性的机制，我们用携带supF基因作为突变靶点的紫外线照射（254 nm）穿梭载体转染XP变异细胞系，允许质粒复制，确定诱导突变的频率和谱，并将结果与之前携带相同靶基因的照射质粒在正常细胞系中复制时获得的结果进行比较[Bredberg, A., Kraemer, K. H. & Seidman, M. M. (1986) 程序。国家。阿卡德。科学。美国 83, 8273-8277]。突变体的频率随剂量呈线性增加，但斜率比正常细胞的斜率陡 5 倍。对supF基因的序列分析表明，XP变异细胞中产生的53个独立突变体中的52个含有碱基取代，其中64个取代中的62个涉及二嘧啶。 28% 的突变涉及 A.T 碱基对，其中大多数突变位于 136 位，即三个 A.T 碱基对的中间位置。 （在正常细胞中，该值仅为 11%。）如果两个细胞系中从 suF 切除病变的比率相等，我们的数据表明 XP 变异细胞比正常细胞更不可能掺入光产物中涉及的 dAMP 相对碱基。如果这种掺入也发生在染色体 DNA 复制过程中，则可以解释 XP 变异细胞在紫外线照射下的超突变性。
Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, we transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the supF gene as a target for mutations, allowed replication of the plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene replicated in a normal cell line [Bredberg, A., Kraemer, K. H. & Seidman, M. M. (1986) Proc. Natl. Acad. Sci. USA 83, 8273-8277]. The frequency of mutants increased linearly with dose, but with a slope 5 times steeper than that seen with normal cells. Sequence analysis of the supF gene showed that 52 of 53 independent mutants generated in the XP variant cells contained base substitutions, with 62 of 64 of the substitutions involving a dipyrimidine. Twenty-eight percent of the mutations involved A.T base pairs, with the majority found at position 136, the middle of a run of three A.T base pairs. (In the normal cells, this value was only 11%.) If the rate of excision of lesions from supF in the two cell lines is equal, our data suggest that XP variant cells are less likely than normal cells to incorporate dAMP opposite bases involved in photo-products. If such incorporation also occurs during replication of chromosomal DNA, this could account for the hypermutability of XP variant cells with UV irradiation.