MECHANISMS OF MUTAGENIC PROCESSING OF DNA DAMAGE

描述：着色性干皮病（XP）患者具有遗传性 易患阳光诱发的皮肤癌，而来自皮肤癌的细胞 这些患者中的大多数已被证明缺乏核苷酸 切除修复 缺乏修复紫外线诱导的DNA损伤可以解释为什么 这些细胞对UV诱导的突变极其敏感。 然而，在这方面， 一类XP患者，命名为变体，在切除中没有缺陷 修复，但他们的细胞表现出更大的敏感性， 紫外线诱导的突变比那些经典的切除修复缺陷XP 患者 紫外线诱导的突变光谱数据表明， XP变体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， 包含照片产品，他们经常犯错误，这些错误是 非常特别的一种 最近的数据，来自体外DNA 比较XP变体和HeLa细胞提取物的复制测定证实， 这是真的。 XP变异细胞的复制能力提取物 代表了第一个也是迄今为止唯一一个 复制复合物，表现出错误倾向绕过DNA损伤。 这些细胞代表了一个独特的机会，研究机制， 正常细胞在复制受损DNA时， 复制中的错误导致突变。 由于突变是因果关系 参与肿瘤转化过程，本研究具有直接 与肿瘤发生研究的相关性。 建议研究 正常细胞提取物绕过许多病变的机制， 从XP变异细胞中分离出来 还建议调查各种 复制蛋白，以确定它们是否可能有助于，或 负责XP变异细胞中的复制缺陷。 具体地说， 建议：1）测试来自正常细胞的细胞提取物是否给出 当复制UV受损时，与HeLa细胞提取物相同的突变谱 模板在体外; 2）扩大目前的数据库紫外线诱导的突变 通过用XP变体细胞提取物复制介导; 3）将 复制基底相对于靶的复制起点 基因，以证实XP变异复制复合物处理光产物 不对称地，以链特异性方式; 4）测试添加是否 RP-A、RF-C、PCNA或pol α在体外校正XP变体缺陷， 并对来自2XP变体的pol δ基因的编码区进行测序 细胞系; 5）制备来自各种XP变体细胞系的提取物， 测试互补性;以及6）通过正常 和XP变体提取物的以下病变放置在特定的网站， 相同的序列背景：T-T二聚体，T-T 6 - 4s， N2-dG-（+）-反式-抗-苯并[a]芘，C8-dG-1-氨基比林，或 C8-dG-2-氨基芴。

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.