LESION-BYPASS DNA POLYMERASES
病变旁路 DNA 聚合酶
基本信息
- 批准号:8170600
- 负责人:
- 金额:$ 0.24万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-07-01 至 2011-06-30
- 项目状态:已结题
- 来源:
- 关键词:Active SitesBindingBypassComplexComputer Retrieval of Information on Scientific Projects DatabaseCytidineDNADNA-Directed DNA PolymeraseDeoxycytidineDeoxyguanosineEnzymesFrequenciesFundingGoalsGrantGuanosineHomologous GeneHumanInstitutionLesionLinkMolecularNucleotidesPolymerasePositioning AttributeProteinsPyrimidinePyrimidinesResearchResearch PersonnelResolutionResourcesSideSolventsSourceSpecificityStagingStructureTimeUnited States National Institutes of HealthWorkadductbasepreferencerat Ran 2 protein
项目摘要
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 enzymes 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.
这个子项目是许多研究子项目中的一个
由NIH/NCRR资助的中心赠款提供的资源。子项目和
研究者(PI)可能从另一个NIH来源获得了主要资金,
因此可以在其他CRISP条目中表示。所列机构为
研究中心,而研究中心不一定是研究者所在的机构。
我们目前正在研究的DinB同系物(Dbh)病变旁路DNA聚合酶从S。solfataricus,并将在明年将我们的工作扩展到人类DinB同源物(Pol kappa)。在接下来的两年里,我们将集中在这些病变旁路聚合酶的两个方面:未受损DNA模板的错误特异性。Dbh聚合酶在遇到特定热点序列时以高频率进行-1移码缺失。这些热点由模板链中的2个或3个嘧啶组成,其5侧接鸟苷。当酶遇到这些热点时,它有50%的时间跳过其中一个模板嘧啶。这种极高的错误率可能与酶对掺入脱氧胞苷的偏好有关(与其他3种核苷酸相比,10倍的偏好)。我们希望了解这些错误产生的结构机制和跳过模板嘧啶和优先将胞苷掺入引物strands.We的序列特异性已经结晶和解决(分子置换)Dbh的结构与DNA复合含有一个跳过的模板碱基在两个不同的位置。在一个位置,正好与活性位点相对,碱基从引物-模板双链体中凸出,在那里它只与溶剂接触,而不与蛋白质接触。在另一个位置,代表在碱基被跳过后两个核苷酸被掺入的阶段,碱基从引物-模板双链体中凸出,在那里它被结合在酶上的小口袋中(见附图)。我们目前正在将这些结构细化到3.0和2.7的高分辨率极限。我们明年的目标是确定共晶结构,其中凸出碱基已被改变为其他3个核苷酸中的每一个(我们目前的结构具有胞苷作为凸出碱基)。这些结构将提供关于酶跳过嘧啶模板碱基的偏好的信息。通过将凸出的碱基放置在模板链的几个不同位置,我们希望了解-1移码错误是如何首先产生,然后传播的。此外,我们计划确定4个输入核苷酸中的每一个与互补模板碱基配对的结构。绕过DNA模板损伤。Dbh最近已被证明有效地绕过脱氧鸟苷的N2-糠基加合物,通过将脱氧胞苷与受损的模板碱基相对。在这项工作的第二年,我们计划通过确定Dbh与含有脱氧鸟苷模板碱基的DNA与各种N2-加合物复合的共晶体结构来研究这种病变旁路的特异性。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Janice D Pata其他文献
Differential gene regulation in Yersinia pestis versus Yersinia pseudotuberculosis: effects of hypoxia and potential role of a plasmid regulator.
鼠疫耶尔森氏菌与假结核耶尔森氏菌的差异基因调控:缺氧的影响和质粒调节剂的潜在作用。
- DOI:
- 发表时间:
2007 - 期刊:
- 影响因子:0
- 作者:
G. Bai;E. Smith;A. Golubov;Janice D Pata;K. McDonough - 通讯作者:
K. McDonough
Janice D Pata的其他文献
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{{ truncateString('Janice D Pata', 18)}}的其他基金
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
7931227 - 财政年份:2009
- 资助金额:
$ 0.24万 - 项目类别:
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
7533198 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
8312533 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
7659644 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
7905151 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Structural and Mechanistic Studies of Error-Prone Polymerases
易错聚合酶的结构和机制研究
- 批准号:
8118977 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Mechanisms of Bacterial DNA Polymerase Replication and Fidelity
细菌 DNA 聚合酶复制和保真度的机制
- 批准号:
8817982 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
Mechanisms of Bacterial DNA Polymerase Replication and Fidelity
细菌 DNA 聚合酶复制和保真度的机制
- 批准号:
9273029 - 财政年份:2008
- 资助金额:
$ 0.24万 - 项目类别:
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