DYNAMIC FLUORESCENCE STUDIES OF DNA/PROTEIN COMPLEXES
DNA/蛋白质复合物的动态荧光研究
基本信息
- 批准号:2022379
- 负责人:
- 金额:$ 25.89万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1992
- 资助国家:美国
- 起止时间:1992-01-15 至 1998-06-30
- 项目状态:已结题
- 来源:
- 关键词:DNA binding protein DNA directed DNA polymerase DNA repair DNA replication active sites chemical binding chemical structure function fluorescence spectrometry fluorescent dye /probe frameshift mutation nucleic acid denaturation nucleic acid sequence protein sequence site directed mutagenesis spleen exonuclease stop flow technique synthetic nucleic acid thermodynamics
项目摘要
Accurate replication of DNA is an essential requirement of all living
organisms, and errors made in copying genetic material can result in a
wide range of disorders. Although DNA is synthesized in vivo with
extremely high fidelity by DNA polymerases, it is not clearly understood
how this fidelity is achieved on a molecular level. The broad, long term
objective of this proposal is to understand how mismatched bases resulting
from polymerase errors are recognized and selectively removed the enzyme.
The proposal will initially focus on the Klenow fragment of DNA Polymerase
I from E. coli, which has served as a model for describing the molecular
basis of DNA replication fidelity, and will subsequently be extended to
polymerases from other organisms. Removal of misincorporated bases by
Klenow fragment appears to involve melting and translocation of the 3' end
of the DNA into a separate editing domain of the enzyme, followed by
exonucleolytic removal of the mismatched base and subsequent return of DNA
to the polymerization domain. During the previous period of support, a
novel solution spectroscopic method was used to make a direct measurement
of DNA bound to the polymerization domain and to the editing domain. It is
proposed to use this method to investigate the structural features of both
the DNA and enzyme that enable mismatched base pairs to be recognized. The
specific aims are: 1. Test the hypothesis that melting of two or more base
pairs is required for transfer of the primer terminus into the editing
domain of Klenow fragment. 2. Determine whether preferential partitioning
of mismatched base pairs into the editing domain is due to increased
melting capacity of the DNA, disfavored binding of DNA to the
polymerization domain, or a combination of these effects. 3. Identify
amino acid residues involved in DNA binding and recognition of mismatched
bases. 4. Measure the rate of transfer of the primer terminus between the
polymerization domain and the editing domain and identify amino acid
residues that facilitate transfer. 5. Determine whether Klenow fragment
suppresses frameshift mutations by rejecting misaligned sequences within
the polymerization domain. 6. Characterize the editing function of other
DNA polymerases m order to test the generality of the principles of
mismatch recognition established for Klenow fragment. Time-resolved
fluorescence anisotropy decay of dansyl-labeled DNA will be used to
measure the distribution of DNA termini bound to the polymerization or
editing domains of Klenow fragment. The DNA and enzyme will be judiciously
modified using oligonucleotide synthesis and site-directed mutagenesis
techniques, respectively, and the effect on the distribution of DNA in
each domain will be determined. Thermodynamic data describing binding of
mismatched DNAs to each domain of the enzyme, and melting of mismatched
DNA sequences, will also be obtained. Stopped-flow fluorescence techniques
will be used to measure the rate of transfer of DNA between domains. The
information obtained from this study will provide insight into the
molecular mechanisms used to suppress mutations during DNA replication.
DNA的精确复制是所有生命的基本要求
生物,在复制遗传物质时发生的错误可能导致
各种各样的疾病。虽然DNA是在体内合成的,
由于DNA聚合酶具有极高的保真度,
这种保真度是如何在分子水平上实现的广泛的,长期的
这项建议的目的是了解错配碱基如何导致
从聚合酶错误中识别并选择性地去除酶。
该提案最初将集中在DNA聚合酶的Klenow片段上
从E。大肠杆菌,它已经作为一个模型,用于描述分子
DNA复制保真度的基础,并将随后扩展到
其他生物的聚合酶。通过以下方法去除错误掺入的碱基:
Klenow片段似乎涉及3'端的解链和易位
将DNA插入酶的一个单独的编辑结构域,然后
错配碱基的核酸外切酶去除和随后的DNA返回
到聚合域。在上一个支助期间,
采用一种新的溶液光谱法直接测定了
结合到聚合结构域和编辑结构域的DNA。是
建议使用这种方法来研究两者的结构特征
能识别错配碱基对的DNA和酶。的
具体目标是:1.检验两个或多个碱基的熔融
将引物末端转移到编辑区需要一对引物
Klenow片段。2.确定是否优先分区
错配碱基对进入编辑结构域是由于增加
DNA的解链能力,不利于DNA与
聚合域,或这些效应的组合。3.识别
参与DNA结合和错配识别的氨基酸残基
基地4.测量引物末端在引物之间的转移速率。
聚合结构域和编辑结构域并鉴定氨基酸
促进转移的残基。5.确定克列诺碎片
抑制移码突变通过拒绝错配序列内
聚合域。6.描述其他编辑功能
DNA聚合酶m为了测试的原则,
错配识别建立Klenow片段。 时间分辨
丹磺酰标记的DNA的荧光各向异性衰减将用于
测量与聚合结合的DNA末端的分布,或
Klenow片段的编辑结构域。DNA和酶将被明智地
使用寡核苷酸合成和定点诱变进行修饰
技术,分别,和对DNA的分布的影响,
每个域都将被确定。描述结合的热力学数据
错配的DNA到酶的每个结构域,和解错配的DNA,
DNA序列,也将获得。停流荧光技术
将用于测量域之间的DNA转移速率。的
从这项研究中获得的信息将提供深入了解
在DNA复制过程中抑制突变的分子机制。
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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David P MILLAR其他文献
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{{ truncateString('David P MILLAR', 18)}}的其他基金
Mechanism/inhibition of RNA binding functions of HIV Rev
HIV Rev RNA 结合功能的机制/抑制
- 批准号:
6821914 - 财政年份:2003
- 资助金额:
$ 25.89万 - 项目类别: