Studies on Molecular Mechanisms of Translesion Synthesis and Carcinogenesis

跨损伤合成及癌变的分子机制研究

• 批准号: 12213070
• 负责人: HANAOKA Fumio
• 金额: $ 43.26万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12213070/
• 关键词: XP variant; translesion synthesis; DNA polymerase; DNA lesions; knockout mice; carcinogenesis; 遺伝子ノックアウト; REV1; DNAポリメラーゼ; DNA修復; 色素性乾皮症; polイータ; DNA複製; 活性酸素; XPVポリメラーゼ; 紫外線

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.

为了了解跨损伤合成的分子机制以及肿瘤发生与跨损伤合成的关系，我们以人DNA聚合酶η（Pol η）为研究对象，对跨损伤合成的各个方面进行了研究，并获得了以下结果.人类Pol η复制未受损DNA的保真度比任何其他研究的模板依赖性DNA聚合酶低得多。另一方面，人Pol η催化通过环合环丁烷二胸腺嘧啶损伤、AAF修饰的鸟嘌呤和顺铂诱导的两个鸟嘌呤之间的链内交联的有效且准确的跨损伤合成。人Pol η基因由11个外显子组成，覆盖了转录起始的整个编码区，基因组DNA分析表明，4个XP-V细胞系中有3个存在纯合突变，1个存在杂合点突变，其中1个为主要等位基因的剪接突变.人Pol η结合模板/引物DNA，而不管TT二聚体。相反，只有当引物的3-末端是位于病变对面的腺苷残基时，才观察到与含有TT二聚体的模板/引物DNA的结合增强。当两个核苷酸已经掺入引物中超过TT二聚体位置时，Pol η-模板/引物DNA复合物不稳定。为了研究XP-V患者皮肤癌发生率高的体内模型，我们通过用neo^r基因替换小鼠ES细胞中的Pol η基因的外显子8来破坏小鼠Pol η基因。对于无效突变纯合的小鼠是可存活的、可繁殖的，并且在它们生命的第一年期间没有显示出任何明显的缺陷。来自突变小鼠的Pol η缺陷型成纤维细胞复制UV损伤DNA的能力降低。Pol η缺陷小鼠在UVB照射后发生皮肤肿瘤，而野生型和杂合子同窝出生的小鼠则没有。

项目成果

DNA binding properties of human DNA polymerase η : Implication for polymerase switching.

人类 DNA 聚合酶的 DNA 结合特性 η：对聚合酶转换的影响。

• 发表时间: 2004
• 期刊: Genes to Cells 9
• 作者: Kusurnoto;R.;Masutani;C.;et al.
• 通讯作者: et al.

Bassett E. et al.: "Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin by human DNA polymerases β and η"Biochemistry. 42. 14197-14206 (2003)

Bassett E. 等人：“通过人类 DNA 聚合酶 β 和 η 延伸错配引物末端的效率”生物化学 42. 14197-14206 (2003)

Yamada, A.et al.: "Detection of reduced RNAsynthesis in UV-irradiated Cockayne syndrome B cells using an isolated nuclear system"Biochim.Biophys.Acta. 1592. 129-134 (2002)

Yamada, A.等人：“使用分离的核系统检测紫外线照射的科凯恩综合征 B 细胞中 RNA 合成减少”Biochim.Biophys.Acta。

Servanit, L.et al.: "A role for DNA polymerase β in mutagenic UV lesions bypass"J.Biol.Chem.. 277. 50046-50053 (2002)

Servanit, L. 等人：“DNA 聚合酶 β 在诱变紫外线损伤绕过中的作用”J.Biol.Chem.. 277. 50046-50053 (2002)

Interaction of hREV1 with three human Y-family DNA polymerases

• DOI: 10.1111/j.1356-9597.2004.00747.x
• 发表时间: 2004-06-01
• 期刊: GENES TO CELLS
• 影响因子: 2.1
• 作者: Ohashi, E;Murakumo, Y;Ohmori, H
• 通讯作者: Ohmori, H