Structural and Mechanistic Studies of Error-Prone Polymerases

描述(由申请人提供)：Y-家族DNA聚合酶通过允许复制继续DNA模板中相反的损伤来帮助细胞耐受DNA损伤。这种跨病变的DNA合成可以是准确的，保留了遗传信息的完整性，也可能容易出错，即使模板链中的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掺入与病变相反的脱氧胞苷核苷酸，准确地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酶通常较少发生碱基替换错误，但它们会以较高的速度进行单碱基缺失突变，即在复制过程中跳过模板碱基。我们使用来自Sulfolobus acidocaldarius的DinB同源基因(DBH)作为DinB类DNA聚合酶的模型。DBH已被证明能准确有效地绕过脱氧鸟苷碱基上的DNA损伤；它表现出强烈的偏好，即使在未受损的DNA上也能结合脱氧胞苷核苷酸；它在特定序列上以极高的比率产生单碱基缺失错误。这个建议的目的是提供一个更完整的了解不同Y家族DNA聚合酶之间的结构差异是如何导致不同的病变旁路活性和DNA复制保真度的。我们的中心假设是，DBH被夸大的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。其具体目的是(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是如何通过与其他蛋白质的相互作用来调节的。我们将使用X射线结晶学、计算和生化方法的组合来解决这些问题。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。与公共卫生相关：人类细胞中多种突变的积累可能会导致癌细胞生长，而细菌中的突变可能会导致抗生素耐药性。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.