Structural and Mechanistic Studies of Error-Prone Polymerases

描述（由申请人提供）：y家族DNA聚合酶通过允许DNA模板中相反病变的复制继续帮助细胞耐受DNA损伤。这种翻译DNA合成可以是准确的，保持遗传信息的完整性，也可以是容易出错的，即使模板链上的DNA损伤后来得到修复，也会在基因组中产生突变。属于DinB亚家族的y家族聚合酶能够通过在病灶对面结合脱氧胞苷核苷酸来精确绕过受损的脱氧鸟苷碱基。与其他类型的y家族聚合酶相比，DinB酶通常会产生更少的碱基替换错误，但它们会产生单碱基缺失突变，即在复制过程中跳过模板碱基。我们使用来自Sulfolobus acidocalarius的DinB同源物（Dbh）作为DinB类DNA聚合酶的模型。Dbh已被证明可以准确有效地绕过脱氧鸟苷碱基的DNA损伤；即使在未损伤的DNA上，它也表现出强烈的结合脱氧胞苷核苷酸的偏好；它在特定序列上产生的单碱基缺失错误率非常高。本提案的目的是提供一个更完整的理解如何在不同的y家族DNA聚合酶之间的结构差异导致不同的病变绕道活性和DNA复制保真度。我们的中心假设是，Dbh的夸张突变特异性和病变旁路活性将使我们更容易识别影响这些活性的结构特征。具体目的是：(1)确定Dbh如何产生单碱基缺失突变，(2)阐明Dbh用于复制受损DNA的机制，以及(3)表征Dbh如何通过与其他蛋白质的相互作用进行调节。我们将结合x射线晶体学、计算和生化方法来解决这些问题。这些研究将有助于我们理解y家族聚合酶如何帮助细胞耐受DNA损伤，以及它们如何将突变引入基因组。公共卫生相关性：人类细胞中多种突变的积累可导致癌细胞生长，而细菌中的突变可导致抗生素耐药性。y家族DNA聚合酶似乎是原核和真核细胞中产生的许多突变的原因。在适当的时候抑制这些聚合酶，可能是防止癌症进展或提高抗菌药物治疗效果的有效方法。

DESCRIPTION (provided by applicant): The Y-family DNA polymerases help cells tolerate DNA damage by allowing replication to continue opposite lesions in the DNA template. This translesion DNA synthesis can be accurate, preserving the integrity of the genetic information, or it can be error-prone, producing a mutation in the genome even if the DNA damage in the template strand is repaired later. The Y-family polymerases that belong to the DinB subfamily are able to bypass damaged deoxyguanosine bases accurately by incorporating deoxycytidine nucleotides opposite the lesion. The DinB enzymes generally make fewer base-substitution errors than other types of Y-family polymerases, yet they make single-base deletion mutations, where a template base is skipped during replication, at a high rate. We are using the DinB homolog (Dbh) from Sulfolobus acidocaldarius as a model for the DinB class of DNA polymerases. Dbh has been demonstrated to accurately and efficiently bypass DNA damage at deoxyguanosine bases; it displays a strong preference for incorporating deoxycytidine nucleotides even on undamaged DNA; and it generates single-base deletion errors at an exceptionally high rate at specific sequences. The objective of this proposal is to provide a more complete understanding of how structural differences among the various Y-family DNA polymerases give rise to differing lesion-bypass activity and DNA replication fidelity. Our central hypothesis is that the exaggerated mutational specificity and lesion-bypass activity of Dbh will allow us to more easily identify the structural features that influence these activities. The specific aims are (1) to determine how Dbh generates single-base deletion mutations, (2) to elucidate the mechanisms Dbh uses to replicate damaged DNA, and (3) to characterize how Dbh is regulated by interactions with other proteins. We will use a combination of X-ray crystallographic, computational and biochemical approaches to address these issues. These studies will contribute to our understanding of how the Y-family polymerases help cells tolerate DNA damage and also how they introduce mutations into the genome. PUBLIC HEALTH RELEVANCE: An accumulation of multiple mutations in human cells can lead to cancerous cell growth, while mutations in bacteria can lead to antibiotic resistance. The Y-family DNA polymerases appear to be responsible for many of the mutations produced in both prokaryotic and eukaryotic cells. Inhibiting these polymerases, at appropriate times, could be a useful way to prevent cancers from progressing or to increase the efficacy of antibacterial drug treatments.