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)不断引起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修复系统修复(移除)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。病变旁路是细胞生存所必需的。 损伤旁路是一种DNA损伤耐受过程，它在复制阻断DNA损伤的情况下复制，而不移除损伤，防止损伤诱导的细胞死亡。DNA损伤耐受过程被细分为两条平行的途径：容易出错的和无错的损伤旁路。容易出错的一种使用称为低保真跨病变DNA聚合酶的特殊蛋白质来在受损的DNA模板上进行直接复制并绕过病变，但代价是突变率增加。无错误的途径绕过了DNA损伤，而不会增加突变率。虽然容易出错的病变旁路已被很好地描述，但对无错误途径的了解较少。无错误的病变搭桥需要同源重组(HR)或DNA交换，但涉及的分子事件尚不清楚。 最近，在芽殖酵母中已鉴定出四个基因，CSM2、PSY3、SHU1和SHU2，它们与无错误损伤旁路有关。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DNA链交换)的调节器，对于无错误的DNA损伤旁路非常重要。 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和功能的研究。本研究目前的目标是：1)确定Shu复合体与不同DNA结构之间的相互作用，2)确定不同组装形式的Shu复合体的结构，3)确定DNA结合形式的Shu复合体的结构。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，来研究舒络合物。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，以及这四个Shu蛋白如何协同促进HR偶联的无错误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(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.