Molecular Mechanism of Error-free DNA Damage Tolerance

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。大多数损伤是由DNA修复系统修复的。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，并对细胞存活构成严重问题。病变旁路通路是细胞存活所必需的。*病变旁路是一种DNA损伤耐受过程，在不去除病变的情况下通过复制阻断DNA病变进行复制，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：易出错和无错误的病变旁路。易出错的一种是使用一种叫做低保真翻译DNA聚合酶的特殊蛋白质，在受损的DNA模板上进行直接复制，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。虽然易发错误的病变旁路已被很好地表征，但对无错误通路的了解较少。无差错病变旁路需要同源重组（HR）或DNA交换，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DNA链交换）的调节剂，对无差错DNA损伤旁路至关重要。*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复合物与不同DNA结构之间的相互作用，2)确定不同组装形式下Shu复合物的结构，3)确定DNA结合形式下Shu复合物的结构。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Shu复合物。所提出的研究将帮助我们确定哪些Shu蛋白操纵DNA结合的特异性，以及四种Shu蛋白如何合作促进hr偶联无错误DNA病变旁路。

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(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.