Molecular Mechanism of Error-free DNA Damage Response

无错误 DNA 损伤反应的分子机制

• 批准号: RGPIN-2019-06165
• 负责人: Ling, Hong
• 金额: $ 2.33万
• 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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738752
• 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 by DNA repair. 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. The DNA damage tolerance process is divided into two parallel pathways: error-prone and error-free lesion bypasses. The error-free pathway bypasses DNA lesions, without increasing the mutation rates. A little knowledge exists for the error-free pathway. Homologous recombination (HR) 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 exchange and important to error-free DNA lesion bypass. In addition, Shu homologs have been identified in fission yeast, C. elegans and humans, indicating a common mechanism has been conserved in eukaryotes. 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) determine the biochemical functions of the Shu complex; 2) determine the structures of the Shu complex in binary (with ATP, or DNA) and ternary forms (with both ATP and DNA) forms; 3). determine the structure of the human Shu homolog SWS1-SWAP1. We will use X-ray crystallography analysis, in combination with molecular biology and biochemistry techniques, to study the tetrameric complex. The proposed studies of the Shu complex will help us to determine which Shu proteins manipulate the specificity of DNA binding and how the Shu proteins cooperate to facilitate HR and error-free DNA lesion bypass.

由于细胞代谢过程或环境条件（如紫外线、电离辐射和化学试剂）不断产生DNA损伤，基于DNA的生物体面临着保持基因组稳定性的巨大挑战。各种DNA损伤反应已经发展到处理DNA损伤和基因组改变；这些反应包括DNA修复和病变绕道。大多数病变（损伤）通过DNA修复。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，并对细胞存活构成严重问题。病变旁路通路是细胞存活所必需的。DNA损伤耐受过程分为两个平行的途径：易出错和无错误的病变旁路。无错误途径绕过DNA损伤，而不增加突变率。对于无错误的路径存在一些知识。同源重组（Homologous recombination， HR）是无差错病变旁路治疗的必要条件，但涉及的分子事件尚不清楚。最近，在出芽酵母中发现了4个基因CSM2、PSY3、SHU1和SHU2参与了无差错病变旁路。这四个基因产物形成了一个稳定的4亚基Shu复合物，这是高效HR所必需的。这些基因中的任何一种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，并招募HR蛋白来促进DNA链转换。因此，Shu复合物是DNA重组或交换的调节剂，对无差错DNA病变旁路至关重要。此外，在裂变酵母、秀丽隐杆线虫和人类中也发现了Shu同源基因，这表明在真核生物中存在一种共同的机制。为了揭示无差错病变旁路的分子机制，我们建议对Shu复合物进行结构-功能研究。本研究目前的目标是：1)确定Shu复合物的生化功能；2)确定Shu配合物的二元（含ATP或DNA）和三元（含ATP和DNA）结构；3).确定人类蜀属同源物SWS1-SWAP1的结构。我们将使用x射线晶体学分析，结合分子生物学和生物化学技术，来研究四聚体复合物。对Shu复合物的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Shu蛋白如何合作促进HR和无差错DNA病变旁路。