Novel Mechanism Ensuring Replication Fidelity

确保复制保真度的新颖机制

• 批准号: 9547584
• 负责人: Guo-Min Li
• 金额: $ 28.8万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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/9547584
• 关键词: Address; Adult; Alpha Cell; Amino Acid Motifs; Biological Assay; 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; Mutation Analysis; N-terminal; Nucleosomes; PWWP Domain; Play; Proteins; Reaction; Recruitment Activity; Role; S phase; System; Technology; Testing; United States; beta-Galactosidase; cancer biomarkers; cancer therapy; chromatin immunoprecipitation; exome sequencing; gel electrophoresis; genome-wide; improved; next generation sequencing; novel; novel marker; public health relevance; screening

 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）维持基因组稳定性的分子机制。MMR通过纠正DNA复制过程中产生的错对，在复制保真度方面发挥着重要作用。错配识别蛋白hMutSα是由hMSH 2和hMSH 6亚基组成的异源二聚体，可以说是MMR中最重要的组成部分。hMSH 6亚基含有PCNA相互作用蛋白基序（称为PIP盒）和在其N端的PWWP结构域。虽然没有明确的功能被分配给PWWP结构域，PIP框以前被认为参与招募hMutSα到错配的DNA。有趣的是，最近的研究表明，PIP盒的缺失仅适度增加突变频率，并且不影响hMutSα染色质定位。令人惊讶的是，我们最近发现MSH 6 PWWP结构域与组蛋白标记H3 K36 me 3物理相互作用，并负责将hMutSα定位于复制的染色质。这些观察结果表明，PCNA-hMSH 6相互作用不是为了hMutSα募集，并且表观遗传H3 K36 me 3组蛋白标记在MMR和基因组稳定性中起关键作用。考虑到H3 K36 me 3在S期的丰度以及PCNA与hMutSα和复制型DNA聚合酶δ和ε的相互作用，我们假设单个核小体中的H3 K36 me 3-hMutSα相互作用决定了相应DNA序列的突变率，并且PCNA-hMutS α相互作用协调了复制叉处的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是通过ChIP-Seq分析确定H3 K36 me 3和hMutSα的全基因组分布和相互作用。具体目标2是研究PCNA-hMutSα相互作用如何调节DNA合成和MMR的分子细节。具体目的3是分别使用外显子组测序分析和诱变试验确定hMutSα与H3 K36 me 3和PCNA相互作用对基因突变的影响。该研究的成功完成不仅将阐明MMR维持基因组稳定性的新机制，还将为癌症检测和治疗提供潜在的新生物标志物。