Molecular Mechanism of Error-free DNA Damage Tolerance

无错误DNA损伤耐受性的分子机制

• 批准号: RGPIN-2014-04473
• 负责人: Ling, Hong
• 金额: $ 3.42万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587833
• 关键词: Molecular; Mechanism; Error; free; DNA

DNA-based organisms face a great challenge of maintaining genomic stability, due to DNA damage that arises constantly by cellular metabolic processes or by environmental conditions (such as UV and ionizing radiation and chemical agents). A variety of DNA damage responses have evolved to deal with DNA damage and genomic alterations; these responses include DNA repair and lesion bypass. Most lesions (damage) are repaired(removed) by DNA repair systems. However, some lesions are resistant to DNA repair, become blocking sites for normal DNA replication, and pose serious problems for cell survival. Lesion bypass pathways are required for cell survival. Lesion bypass is a DNA-damage tolerance process that replicates through the replication-blocking DNA lesions without removal of the lesions and prevents damage-induced cell death. The DNA damage tolerance process is subdivided into two parallel pathways: error-prone and error-free lesion bypasses. The error-prone one uses special proteins called low-fidelity translesion DNA polymerases to carry out direct replication over the damaged DNA template and bypass lesions, but at a cost of increased mutation rates. The error-free pathway bypasses DNA lesions without increasing the mutation rates. While error-prone lesion bypass has been well characterized, less knowledge exists for the error-free pathway. Homologous recombination (HR) or DNA exchange is required for error-free lesion bypass, but the molecular events involved are not clear. Recently, four genes in budding yeast, CSM2, PSY3, SHU1 and SHU2, have been identified that are involved in the error-free lesion bypass. The four gene products form a stable, 4-subunit Shu complex that is required for efficient HR. Inactivation of any of these genes makes yeast cells more sensitive to DNA damage agents. The Shu complex binds both single- and double-stranded DNA and appears to recruit HR proteins to facilitate DNA strand switching. Thus, the Shu complex is a regulator of DNA recombination (or DNA strand exchange) and important to error-free DNA lesion bypass. To reveal the molecular mechanism of error-free lesion bypass, we propose to conduct a structure-function study of the Shu complex. The current goals of this study are: 1) to determine the interactions between the Shu complex and different DNA structures, 2) to determine the structures of the Shu complex in different assembly forms, 3) to determine the structures of the Shu complex in DNA-bound forms. We will use X-ray crystallography analysis, in combination with molecular biology and biochemistry techniques, to study the Shu complex. The proposed studies will help us to determine which Shu proteins manipulate the specificity of DNA binding and how the four Shu proteins cooperate to facilitate HR-coupled error-free DNA lesion bypass.

基于DNA的生物体面临着维持基因组稳定性的巨大挑战，这是由于细胞代谢过程或环境条件（如紫外线和电离辐射和化学试剂）不断引起的DNA损伤。已经进化出多种DNA损伤反应来处理DNA损伤和基因组改变;这些反应包括DNA修复和病变旁路。大多数损伤（损伤）是通过DNA修复系统修复（去除）的。然而，一些病变对DNA修复具有抗性，成为正常DNA复制的阻断位点，并对细胞存活造成严重问题。病变旁路途径是细胞存活所必需的。 损伤绕过是一种DNA损伤耐受过程，它通过复制阻断的DNA损伤进行复制，而不去除损伤，并防止损伤诱导的细胞死亡。DNA损伤耐受过程被细分为两个平行的途径：易错和无错损伤旁路。容易出错的一种使用称为低保真度跨病变DNA聚合酶的特殊蛋白质在受损的DNA模板上进行直接复制并绕过病变，但代价是增加突变率。无错误途径绕过DNA损伤而不增加突变率。虽然易错病变旁路已得到很好的表征，但对无错通路的了解较少。同源重组（HR）或DNA交换是无错误病变旁路所必需的，但涉及的分子事件尚不清楚。 最近，已经鉴定了芽殖酵母中的四个基因，CSM 2、PSY 3、SHU 1和SHU 2，它们参与了无错误的病变旁路。这四个基因产物形成一个稳定的4亚基Shu复合体，这是高效HR所必需的。这些基因中任何一个的失活都会使酵母细胞对DNA损伤剂更敏感。Shu复合物结合单链和双链DNA，并且似乎招募HR蛋白以促进DNA链切换。因此，Shu复合物是DNA重组（或DNA链交换）的调节剂，并且对于无错误的DNA损伤绕过是重要的。 为了揭示无差错病变旁路的分子机制，我们建议进行结构-功能研究的舒复合物。本研究的目的是：1）确定Shu复合物与不同DNA结构之间的相互作用; 2）确定Shu复合物在不同组装形式下的结构; 3）确定Shu复合物在DNA结合形式下的结构。我们将利用X射线晶体学分析，结合分子生物学和生物化学技术，对舒氏配合物进行研究。这些研究将帮助我们确定哪些Shu蛋白操纵DNA结合的特异性，以及四种Shu蛋白如何合作以促进HR偶联的无错误DNA损伤旁路。