Novel Mechanism Ensuring Replication Fidelity

确保复制保真度的新颖机制

基本信息

批准号：
9029015
负责人：
Guo-Min Li
金额：
$ 32.59万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9029015
关键词：
Address Adult Affect Amino Acid Motifs Biological Assay Biological Markers Boxing Cancer Detection Cancer Etiology Cells Cessation of life ChIP-seq Chromatin Color Colorectal Cancer Complement DNA DNA Repair DNA Sequence DNA biosynthesis DNA replication fork DNA-Directed DNA Polymerase Disease Ensure Epigenetic Process Frequencies Gene Mutation Genes Genome Genome Stability Goals Histones In Vitro Individual MSH6 gene Maintenance Mismatch Repair Molecular Mutagenesis Mutation N-terminal Nucleosomes PWWP Domain Play Proteins Reaction Recruitment Activity Research Role S Phase Sequence Analysis System Technology Testing United States beta-Galactosidase cancer therapy chromatin immunoprecipitation exome sequencing gel electrophoresis genome-wide improved next generation sequencing novel novel marker public health relevance screening

项目摘要

DESCRIPTION (provided by applicant) The long-term goal of this project is to understand the molecular mechanism by which DNA mismatch repair (MMR) maintains to genomic stability. MMR plays an important role in replication fidelity by correcting mispairs generated during DNA replication. Mismatch recognition protein hMutSα, a heterodimer consisting of hMSH2 and hMSH6 subunits, is arguably the most important component in MMR. The hMSH6 subunit contains a PCNA interaction protein motif (called PIP box) and a PWWP domain at its N-terminus. While no clear function has been assigned to the PWWP domain, the PIP box was previously thought to be involved in recruiting hMutSα to mismatched DNA. Interestingly, recent studies have shown that depletion of the PIP box only moderately increases mutation frequencies, and does not affect hMutSα chromatin localization. Surprisingly, we show recently that the MSH6 PWWP domain physically interacts with histone mark H3K36me3 and is responsible for localizing hMutSα to replicating chromatin. These observations suggest that the PCNA-hMSH6 interaction is not for hMutSα recruitment and that the epigenetic H3K36me3 histone mark plays a critical role in MMR and genomic stability. Given the abundance of H3K36me3 in S phase and that PCNA interacts with both hMutSα and replicative DNA polymerases δ and ε, we hypothesize that the H3K36me3-hMutSα interaction in individual nucleosomes determines mutation rates in the corresponding DNA sequences, and that the PCNA-hMutSα interaction coordinates the DNA replication and MMR reactions at the replication fork. To test these hypotheses, three specific aims are proposed. Specific aim 1 is to determine genome-wide distributions of and interactions between H3K36me3 and hMutSα by ChIP- Seq analysis. Specific aim 2 is to study molecular details as to how the PCNA-hMutSα interaction regulates DNA synthesis and MMR. Specific aim 3 is to determine the impact of hMutSα interactions with H3K36me3 and PCNA on gene mutations using Exome-Sequencing analysis and a mutagenesis assay, respectively. A successful completion of the proposed study will not only elucidate novel mechanisms by which MMR maintains genome stability, but also provide potentially new biomarkers for cancer detection and therapy.

描述（由申请人提供）该项目的长期目标是了解 DNA 错配修复 (MMR) 通过纠正 DNA 复制过程中产生的错配来维持基因组稳定性的分子机制。由 hMSH2 和 hMSH6 亚基组成，可以说是 MMR 中最重要的组成部分。 hMSH6 亚基包含 PCNA 相互作用蛋白基序（称为 PIP 盒）和 PWWP 结构域。虽然 PWWP 结构域没有明确的功能，但之前认为 PIP 盒参与将 hMutSα 招募到不匹配的 DNA 中，但最近的研究表明，PIP 盒的耗尽只会适度增加突变。令人惊讶的是，我们最近发现 MSH6 PWWP 结构域与组蛋白标记 H3K36me3 发生物理相互作用并负责。这些观察结果表明，PCNA-hMSH6 相互作用并非用于 hMutSα 募集，而且考虑到 S 期 H3K36me3 的丰度，表观遗传 H3K36me3 组蛋白标记在 MMR 和基因组稳定性中发挥着关键作用。 hMutSα 和复制 DNA 聚合酶 δ 和 ε，我们发现单个核小体中的 H3K36me3-hMutSα 相互作用决定了相应 DNA 序列坐标中的突变率，并且 PCNA-hMutSα 相互作用协调复制叉处的 DNA 复制和 MMR 反应为了检验这些假设，提出了三个具体目标具体目标 1。具体目标 2 是通过 ChIP-Seq 分析确定 H3K36me3 和 hMutSα 的全基因组分布和相互作用。 PCNA-hMutSα 相互作用如何调节 DNA 合成和 MMR。具体目标 3 是分别使用外显子组测序分析和诱变测定确定 hMutSα 与 H3K36me3 和 PCNA 相互作用对基因突变的影响。不仅阐明了 MMR 维持基因组稳定性的新机制，而且还为癌症检测和治疗提供了潜在的新生物标志物。