REGULATING GENOME FIDELITY AND CANCER PROGRESSION

调节基因组保真度和癌症进展

• 批准号: 8637495
• 负责人: ROBERT A BAMBARA
• 金额: $ 16.69万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8637495
• 关键词: Acetylation; Acetylesterase; Acetyltransferase; Aging; Base Excision Repairs; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Preservation; Cell Aging; Cell Culture System; Cells; Cleaved cell; Complex; DNA; DNA Primers; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA repair protein; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Deacetylase; Defect; EP300 gene; Environmental Risk Factor; Enzymes; Epigenetic Process; Equilibrium; Eukaryotic Cell; Excision; Flap Endonucleases; Genetic; Genome; Genome Stability; Goals; Growth; Health; Human; Human Genome; In Vitro; Individual; Investigation; Knowledge; Length; Life; Ligation; Lysine; Mass Spectrum Analysis; Measures; Modification; Mutagenesis; Mutation; Nature; Nuclear; Nucleotides; Okazaki fragments; Outcome; Pathway interactions; Plasmids; Polymerase; Post-Translational Protein Processing; Probability; Process; Property; Protein Acetylation; Proteins; RNA; RNA replication; RTH-1 Nuclease; Regulation; Replication Error; Reporter; Role; Saccharomyces cerevisiae; Site; Stress; Surgical Flaps; System; Testing; Ursidae Family; Work; Yeasts; base; carcinogenesis; design; histone acetyltransferase; in vivo; mutant; nuclease; protein function; public health relevance; reconstitution; repaired; replication factor A; synthetic protein; tool; tumor progression; yeast genetics

DESCRIPTION (provided by applicant): Human cells respond to endogenous and exogenous stresses by using post translational modification as a fundamental tool to control protein functions. This regulation controls genome stability and DNA repair in ways that either promote or suppress carcinogenesis. We have evidence that protein acetylation specifically adjusts the fidelity of DNA replication and repair. One strand of the DNA genome is made discontinuously, whereby Okazaki fragments about 150 nucleotides long are synthesized and joined. Similar mechanisms, and mostly the same enzymes, are employed by the frequently-utilized base excision repair system. For replication, RNA/DNA primer of each fragment, made by the error-prone DNA polymerase ?, is raised into a flap by synthesis from the adjacent fragment. For repair, a damaged part of DNA is also made into a flap. In both cases the flap is removed by flap endonuclease (FEN1) and then adjacent segments are joined. Our previous reconstitution analyses showed that most replication/repair flaps were removed while short, so that a minimal-length synthesis patch was made before fragment joining. However, some flaps became long, and a large patch was replaced, requiring the additional function of the Dna2 nuclease for long flap cleavage. Why should two pathways, long and short flap creation and removal, have evolved? We have evidence that they represent a fundamental regulation process based on these observations: Acetylation of human FEN1 by the histone acetyltransferase p300 lowers nuclease activity. Acetylation of Dna2 nuclease by p300 greatly enhances cleavage activity. Acetylation stimulates DNA polymerases that displace the flaps. Together, these effects suggest a regulation in which longer flaps are created, properly processed by Dna2, but joined only after a long patch is replaced because of the lower FEN1 activity. Such regulation would cause replication/repair proteins to replace a longer patch, assuring Okazaki primer removal in replication and complete damage removal in repair. The regulation would balance fidelity with efficiency. Preliminary evidence in yeast having an enhanced error-prone DNA polymerase ?, showing that deletion of a major protein acetylase decreases fidelity of DNA replication, supports this hypothesis. Our proposal has two key components: One, the enzymatic changes caused by acetylation of replication/repair proteins will be defined for individual proteins, interacting protein partners, and reconstitutions of the replication and repair pathways. Results should reveal whether acetylation changes protein function consistent with the original hypotheses, or suggest alternative explanations. Two, genetic and cell biological approaches will be applied to determine the effects of replication/repair protein acetylation in the cell. We ill specifically establish whether acetylation is used to regulate patch replacement length in replication/repair, and whether that regulation alters the fidelity of DNA synthesis.

描述（由申请人提供）：人细胞通过使用翻译后修饰作为控制蛋白质功能的基本工具来响应内源性和外源性应激。这种调节以促进或抑制致癌作用的方式控制基因组稳定性和DNA修复。我们有证据表明，蛋白质乙酰化特异性地调节DNA复制和修复的保真度。DNA基因组的一条链是不连续的，由此合成并连接约150个核苷酸长的冈崎片段。常用的碱基切除修复系统采用类似的机制，并且大多数是相同的酶。对于复制，每个片段的RNA/DNA引物，由易错DNA聚合酶？，通过与相邻片段的合成而升高成瓣。为了修复，DNA的受损部分也被制成皮瓣。在这两种情况下，通过瓣核酸内切酶（FEN 1）去除瓣，然后连接相邻的片段。我们以前的重建分析表明，大多数复制/修复皮瓣被删除，而短，使最小长度的合成补丁之前，片段连接。然而，一些瓣变得很长，并且大的补丁被替换，需要DNA 2核酸酶的额外功能来切割长瓣。为什么会进化出两种途径，长瓣和短瓣的产生和去除？我们有证据表明，他们代表了一个基本的调控过程的基础上，这些观察：乙酰化的人FEN 1的组蛋白乙酰转移酶p300降低核酸酶的活性。DNA 2核酸酶被p300乙酰化大大增强切割活性。乙酰化作用刺激DNA聚合酶，使皮瓣移位。总之，这些效应表明了一种调节，其中较长的皮瓣被创建，由Dna 2正确处理，但由于FEN 1活性较低，只有在长的补丁被替换后才连接。这样的调节会导致复制/修复蛋白取代较长的补丁，确保冈崎引物在复制中被去除，并在修复中完全去除损伤。这项规定将在忠诚与效率之间取得平衡。酵母中具有增强的易错DNA聚合酶的初步证据，表明主要蛋白乙酰化酶的缺失降低了DNA复制的保真度，支持了这一假设。我们的建议有两个关键组成部分：一，由复制/修复蛋白的乙酰化引起的酶的变化将被定义为单个蛋白质，相互作用的蛋白质伴侣，以及复制和修复途径的重建。结果应该揭示乙酰化是否改变了蛋白质功能，与最初的假设一致，或者提出替代解释。第二，将应用遗传学和细胞生物学方法来确定细胞中复制/修复蛋白乙酰化的影响。我们将具体确定乙酰化是否用于调节复制/修复中的补丁替换长度，以及这种调节是否改变DNA合成的保真度。