LESION-BYPASS DNA POLYMERASES

病变旁路 DNA 聚合酶

• 批准号: 7726271
• 负责人: Janice D Pata
• 金额: $ 1.04万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726271
• 关键词: Active Sites; Binding; Bypass; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytidine; DNA; DNA-Directed DNA Polymerase; Deoxycytidine; Deoxyguanosine; Enzymes; Frequencies; Funding; Goals; Grant; Guanosine; Homologous Gene; Human; Institution; Lesion; Link; Molecular; Nucleotides; Polymerase; Positioning Attribute; Proteins; Pyrimidine; Pyrimidines; Rate; Research; Research Personnel; Resolution; Resources; Side; Solvents; Source; Specificity; Staging; Structure; Time; United States National Institutes of Health; Work; adduct; base; pol genes; preference; rat Ran 2 protein

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are currently studying in the DinB homolog (Dbh) lesion bypass DNA polymerase from S. solfataricus and will extend our work to the human DinB homolog (Pol kappa) within the next year. In the next two years we will focus on two aspects of these lesion-bypass polymerases: a. Error specificity on undamaged DNA templates. The Dbh polymerase makes -1 frameshift deletions at a high frequency when it encounters specific ¿¿¿¿¿¿hotspot¿¿¿¿¿¿ sequences. These hotspots consist of a run of 2 or 3 pyrimidines in the template strand flanked on the 5¿¿¿¿¿¿ side by a guanosine. When the enzyme encounters these hotspots, it skips one of the templating pyrimidines 50% of the time. This tremendously high error rate is probably linked to the enzyme¿¿¿¿¿¿s preference for incorporating deoxycytidine (10-fold preference compared to the other 3 nucleotides). We want to understand the structural mechanism by which these errors are generated and the sequence specificity for skipping template pyrimidines and for preferentially incorporating cytidine into the primer strand. We have crystallized and solved (by molecular replacement) the structures of Dbh complexed with DNA containing a skipped template base at two different positions. In one position, just opposite the active site, the base is bulged out of the primer-template duplex where it is only in contact with solvent, not protein. In the other position, representing a stage where two nucleotides have been incorporated after the base was skipped, the base is bulged out of the primer-template duplex where it is bound in a small pocket on the enzyme (see attached figure). We are currently refining these structures to high resolution limits of 3.0 and 2.7 . Our goals over the next year are to determine co-crystal structures where the bulged base has been changed to each of the other 3 nucleotides (our current structures have cytidine as the bulged base). These structures will provide information about the preference for the enzyme skipping pyrimidine template bases. By placing the bulged base at several different positions in the template strand, we hope to understand how -1 frameshift mistakes are first generated, and then propagated. Additionally, we plan to determine structures with each of the 4 incoming nucleotides pairing with a complementary templating base. b. Bypass of DNA template lesions. Dbh has recently been shown to bypass an N2-furfuryl adduct of deoxyguanosine efficiently by incorporating deoxycytidine opposite the damaged template base. In the second year of this work, we plan to investigate the specificity of the bypass of this lesion by determining co-crystal structures of Dbh complexed with DNA containing deoxyguanosine template bases with various N2-adducts.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 我们目前正在研究Solfataricus的Dinb同源物（DBH）病变旁路DNA聚合酶，并将在明年将我们的工作扩展到人类Dinb同源物（Pol Kappa）。在接下来的两年中，我们将重点介绍这些病变 - 抗原聚合酶的两个方面： 一个。未切割的DNA模板上的误差特异性。当DBH聚合酶遇到特定的ood»`````````````````````````当这些热点由模板链中的2或3个嘧啶的运行组成，该模板链侧面是鸟嘌呤的侧面。当酶遇到这些热点时，它会在50％的时间跳过模板嘧啶之一。这种极高的错误率可能与酶€€»»偏爱脱氧胞苷（与其他3个核苷酸相比10倍）有关。我们想了解产生这些误差的结构机制，以及跳过模板吡imi碱的序列特异性，并优先将胞苷增加到引物链中。 我们已经结晶并求解（通过分子替代）与在两个不同位置的DNA复合的DBH结构。在一个位置，就在活动位点对面的一个位置，将底座膨胀在质板双链体中，仅与溶剂而不是蛋白质接触。在另一个位置，代表一个阶段，在跳过底座后已掺入了两个核苷酸，将底座从引物 - 板双链体中膨胀，其中它在酶上的小袋中结合在一起（请参阅附件图）。我们目前正在将这些结构完善到3.0和2.7的高分辨率限制。 我们明年的目标是确定块状底座已更改为其他3个核苷酸的共晶结构（我们当前的结构具有胞苷作为块状碱基）。这些结构将提供有关酶跳过嘧啶模板碱基的偏好的信息。通过将块底底座放置在模板链中的几个不同位置，我们希望了解如何首先生成-1移码错误，然后再传播。此外，我们计划通过与完整的模板基础配对的4个传入的核苷酸中的每一个确定结构。 b。 DNA模板病变的旁路。最近，DBH通过与受损的模板基础相对的脱氧胞替丁掺入有效地绕过了脱氧鸟苷的N2毛增合蛋白加合物。在这项工作的第二年中，我们计划通过确定与含有各种N2 adducts的DNA复合的DNA复合DNA的DBH的共晶体结构来研究该病变的旁路特异性。