Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light.
Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light.
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着色性干皮病变异细胞提取物对光产物的异常且容易出错的旁路导致了紫外线诱导的突变类型的极端链偏差。
DOI：
10.1128/mcb.19.1.147
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发表时间：
1999
影响因子：
5.3
通讯作者：
McCormick,JJ
中科院分区：
文献类型：
--
作者：
McGregor,WG;Wei,D;Maher,VM;McCormick,JJ
着色性干皮病 (XP) 是一种罕见的遗传性疾病，其特征是对阳光诱发的皮肤癌的易感性大大增加。大多数患者的细胞在核苷酸切除修复方面存在缺陷。然而，来自一组 XP 变体患者的细胞表现出正常修复，但复制含有紫外线光产物的 DNA 的速度异常缓慢。 XP 变异细胞中紫外线辐射诱导突变的频率明显高于正常人类细胞。此外，紫外线诱发的突变类型与正常突变有很大不同。 30% 的碱基取代不是由 C→T 转换组成，而是由 C→A 颠换组成，所有这些都源自位于一条链中的光产物。涉及另一条链中胞嘧啶的突变几乎都是 C→T 转变。百分之四十五的取代涉及胸腺嘧啶，并且大多数是颠换。为了检验紫外线超突变性和异常突变谱是由 DNA 中光产物的异常旁路引起的假设，我们将 XP 变异细胞的提取物与 HeLa 细胞和成纤维细胞株 MSU-1.2 的提取物进行了复制紫外线照射的 I M13 噬菌体的能力。 M13模板包含直接位于目标基因lacZα左侧或右侧的猿猴病毒40复制起点，从而定义了DNA复制的前导链和滞后链的模板。对于 XP 变体细胞提取物，将复制减少至对照值的~37% 只需要每个模板 1 个光产物，但对于 HeLa 或 MSU-1.2 细胞提取物则需要~2.2 个光产物。 XP 变异细胞提取物诱导的突变频率是 HeLa 或 MSU-1.2 细胞提取物的四倍。对于 XP 变体细胞提取物，使用任一 M13 模板，C→A 颠换的比例高达 43%，并且来自位于模板中用于前导链合成的光产物；对于 HeLa 或 MSU-1.2 细胞提取物，该值仅为 5%，并且这些值来自任一链中的光产物。 XP 变体提取物中，26% 的取代涉及胸腺嘧啶，并且几乎全部都是 T→A 颠换。 XP 变异细胞系中 DNA 聚合酶 δ 催化亚基编码区的序列分析揭示了两种多态性，但这些并不能解释旁路保真度降低的原因。我们的数据表明，XP 变异细胞的 UV 超突变性是由于旁路保真度降低所致，并且与正常细胞不同，前导链模板中涉及胞嘧啶的光产物的旁路与滞后链中光产物的旁路显着不同。
Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene,lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ∼37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ∼2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.