MECHANISMS OF MUTAGENIC PROCESSING OF DNA DAMAGE

描述：色素性干皮病(XP)患者有遗传性 对阳光诱导的皮肤癌的易感性，以及来自 这些患者中的大多数被证明是核苷酸缺乏的。 切除修复。紫外线诱导的DNA损伤缺乏修复可以解释为什么 这些细胞对紫外线诱导的突变极其敏感。然而， 一类XP患者，被指定为变种，在切除方面并不存在缺陷 修复，然而他们的细胞表现出对 紫外线诱导的突变比经典的切除修复缺陷XP的突变 病人。来自紫外线诱导突变光谱的数据表明，癌症 XP变异患者的易感性是由于他们的细胞具有 有缺陷的DNA复制复合体，所以当它们复制DNA时 含有感光产品的人经常会犯错误，这些错误是 一种非常独特的。最近的数据，来自一种体外DNA 比较XP变异体和HeLa细胞提取物的复制分析，证实 这是真的。这种从XP变异细胞中提取的具有复制能力的提取物 代表了人类dna的第一个，也是迄今为止唯一的例子。 复制复合体，展示了容易出错的绕过DNA损伤的方法。 这些细胞代表了一个独特的机会来研究机制，通过 哪些正常细胞在复制受损的DNA时达到保真度，以及它们是如何 复制中的错误会导致突变。因为突变是因果关系 参与肿瘤转化过程，本研究具有直接意义 与肿瘤发生学研究的相关性。建议研究一下 正常细胞提取液对多种病变的旁路机制 来自XP变异细胞。还建议调查各种不同的 复制蛋白，以确定它们是否可能对 负责XP变异细胞中的复制缺陷。具体来说， 建议：1)测试从正常细胞中提取的细胞是否具有 与HeLa细胞在复制紫外线损伤时提取的突变谱相同 体外模板；2)扩展现有的紫外线诱变数据库 通过XP变异体细胞提取物进行复制；3)切换 复制底物相对于靶的复制起点 基因，以确认XP变体复制复合体处理感光产品 不对称地，以链特定的方式；4)测试添加是否 Rp-A、Rf-C、增殖细胞核抗原或Polα在体外纠正XP变体缺陷， 并对来自2XP变异体的polDelta基因的编码区进行了测序 细胞系；5)制备各种XP变异细胞系的提取物， 测试互补性；6)按正常方式检查旁路参数 以及以下病变的XP变体提取液，特定位置放置在 相同的序列背景：T-T二聚体，T-T 6-4S， N_2-DG-(+)-反式反[a]苯并[a]芘，C8-DG-1-氨基比林，或 C8-DG-2-氨基荧烯。

DESCRIPTION: Xeroderma pigmentosum (XP) patients have an inherited predisposition to sunlight-induced skin cancer, and the cells from the majority of these patients have been shown to be deficient in nucleotide excision repair. Lack of repair of UV-induced DNA damage can explain why these cells are extremely sensitive to mutations induced by UV. However, one class of XP patients, designated variant, are not deficient in excision repair, and yet their cells exhibit an even greater sensitivity to UV-induced mutations than those of classic excision repair-deficient XP patients. Data from UV-induced mutational spectra suggest that the cancer predisposition of XP variant patients results from their cells having a defective DNA replication complex, so that when they replicate DNA containing photoproducts they make frequent errors, and these errors are of a very distinctive kind. Recent data, derived from an in vitro DNA replication assay comparing XP variant and HeLa cell extracts, confirm that this is true. This replication-competent extract from XP variant cells represents the first and, as yet, the only example of a human DNA replication complex that demonstrates error-prone bypass of DNA damage. These cells represent a unique opportunity to investigate the mechanisms by which normal cells achieve fidelity when they replicate damaged DNA, and how errors in replication lead to mutations. Since mutations are causally involved in the process of neoplastic transformation, this study has direct relevance to the study of tumorigenesis. It is proposed to study the mechanism of bypass of a number of lesions by extracts from normal cells and from XP variant cells. It is also proposed to investigate a variety of replication proteins to determine if they may contribute to, or be responsible for, the replication defect in XP variant cells. Specifically, it is proposed: 1) to test whether cell extracts from normal cells give the same mutation spectrum as HeLa cell extracts when replication UV damaged templates in vitro; 2) to expand a current database of UV-induced mutations mediated by replication with XP variant cell extracts; 3) to switch the origin of replication of the replication substrate relative to the target gene, to confirm that XP variant replication complex processes photoproducts asymmetrically, in a strand-specific manner; 4) to test whether the addition of RP-A, RF-C, PCNA, or pol alpha correct the XP variant defect in vitro, and to sequence the coding region of the pol delta gene from 2 XP variant cell lines; 5) to prepare extracts from a variety of XP variant cell lines, to test for complementation; and 6) to examine bypass parameters by normal and XP variant extracts of the following lesions placed site-specifically in the same sequence context: T-T dimers, T-T 6-4s, N2-dG-(+)-trans-anti-benzo[a]pyrene, C8-dG-1-aminopyrine, or C8-dG-2-aminofluorene.