Molecular Mechanism of Error-free DNA Damage Response

*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)不断造成DNA损伤，以DNA为基础的生物面临着维持基因组稳定的巨大挑战。各种DNA损伤反应已经进化到处理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修复修复的。然而，一些病变对DNA修复具有抵抗力，成为正常DNA复制的阻断部位，并对细胞生存构成严重问题。损伤旁路是细胞生存所必需的。*DNA损伤耐受过程分为两条平行的路径：易出错和无错误损伤旁路。无错误的途径绕过了DNA损伤，而不会增加突变率。对于无错误的路径，存在一些知识。无错误的病变旁路需要同源重组(HR)，但涉及的分子事件尚不清楚。*最近，在发芽酵母中已鉴定出四个基因，CSM2、PSY3、SHU1和SHU2，它们参与了无错误的病变旁路。这四种基因产物形成了一个稳定的4亚单位Shu复合体，这是有效的HR所必需的。这些基因中的任何一个的失活都会使酵母细胞对DNA损伤剂更加敏感。Shu复合体结合单链和双链DNA，并似乎招募HR蛋白来促进DNA链切换。因此，Shu复合体是DNA重组或交换的调节因子，对无错误的DNA损伤旁路很重要。此外，在分裂酵母、线虫和人类中也发现了Shu同源物，这表明在真核生物中存在一种共同的机制。*为了揭示无错误病变旁路的分子机制，我们建议对舒群复合体进行结构-功能研究。本研究目前的目标是：1)确定舒类复合体的生化功能；2)确定舒类复合体的二元型(含ATP或DNA)和三元型(含ATP和DNA)的结构；3)。确定人类Shu同源基因SWS1-SWAP1的结构。我们将使用X射线结晶学分析，结合分子生物学和生物化学技术，来研究四聚体络合物。对Shu复合体的拟议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蛋白如何协作以促进HR和无错误的DNA损伤旁路。

***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.**