Studies on Molecular Mechanisms of Translesion Synthesis and Carcinogenesis

为了了解翻译转化合成的分子机制以及致癌与翻译转化合成之间的关系，我们对翻译转化合成的各个方面进行了研究，并特别参考了人类DNA聚合酶η (Pol η)，得到了以下结果：人类Pol η复制未损伤DNA的保真度远低于其他模板依赖性DNA聚合酶。另一方面，人类Pol η催化了高效准确的翻译合成，通过环-辛环丁烷二胸腺嘧啶病变，aaf修饰的鸟嘌呤，以及顺铂诱导的两种鸟嘌呤之间的链内交联。人类Pol η基因由11个外显子组成，覆盖了整个转录起始编码区，基因组DNA分析表明，4株XP-V细胞株中有3株为纯合突变，1株为杂合点突变，其中1株为主等位基因的剪接突变。人类Pol η结合模板/引物dna，与TT二聚体无关。相反，只有当引物的3端是位于病灶对面的腺苷残基时，才会观察到与含有TT二聚体的模板/引物dna的增强结合。当两个核苷酸在TT二聚体以外的位置被纳入引物时，Pol -模板/引物DNA复合物不稳定。为了建立一个良好的体内模型来研究XP-V患者皮肤癌变的高发生率，我们通过用neo^r基因取代8外显子来破坏小鼠ES细胞中的Pol η基因。零突变纯合子的小鼠是可存活的，可生育的，并且在它们生命的第一年没有表现出任何明显的缺陷。来自突变小鼠的Pol - η缺陷成纤维细胞复制紫外线损伤DNA的能力降低。Pol - η缺乏小鼠在uvb照射后出现皮肤肿瘤，而野生型和杂合的幼崽则没有。

In order to understand the molecular mechanisms of translesion synthesis and the relationships between carcinogenesis and translesion synthesis, we studied various aspects of translesion synthesis with special reference on human DNA polymerase η (Pol η) and obtained following results.1. Human Pol η copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. On the other hand, human Pol η catalyzes efficient and accurate translesion synthesis past cic-syn cyclobutane di-thymine lesions, AAF-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines.2. Human Pol η gene consists of 11 exons covering the entire coding region of the transcription-initiation, and the genomic DNA analysis suggested that three of four XP-V cell lines have homozygous mutations, and one of them have heterozygous point mutations, one of which is a splice mutation of the major allele.3. Human Pol η binds template/primer DNAs regardless of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the Pol η-template/primer DNA complex is destabilized.4. In order to make a good in vivo model to study the high incidence of skin carcinogenesis in XP-V patients, we disrupted the mouse Pol η gene in ES cells by replacing exon 8 with the neo^r gene. Mice that are homozygous for the null mutation are viable, fertile and did not show any obvious defects during their first year of life. Pol η-deficient fibroblasts derived from the mutant mice have reduced capacity to replicate UV-damaged DNA. Pol η-deficient mice developed skin tumors after UVB-irradiation, while wild-type and heterozygous littermates did not.