REGULATING GENOME FIDELITY AND CANCER PROGRESSION

描述(申请人提供)：人类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压力。这种调控以促进或抑制癌变的方式控制着基因组的稳定性和DNA修复。我们有证据表明，蛋白质乙酰化专门调节DNA复制和修复的保真度。DNA基因组的一条链是不连续的，由此合成并连接大约150个核苷酸长的冈崎片段。经常使用的碱基切除修复系统使用类似的机制，并且大多使用相同的酶。为了复制，每个片段的RNA/DNA引物由容易出错的DNA聚合酶？制造，通过合成相邻的片段而形成一个瓣状。为了进行修复，DNA的受损部分也会被制成一个瓣。在这两种情况下，用FEN1(FEN1)将皮瓣移除，然后将相邻的部分连接起来。我们先前的重建分析表明，大多数复制/修复瓣在短时间内被移除，因此在片段连接之前进行了最小长度的合成补丁。然而，一些皮瓣变长了，一个大补丁被替换了，这需要DNA2核酸酶的额外功能来进行长皮瓣切割。为什么应该进化出两条路径，长的和短的皮瓣生成和移除？我们有证据表明，基于这些观察，它们代表了一个基本的调控过程：组蛋白乙酰转移酶p300对人FEN1的乙酰化降低了核酸酶的活性。P300对DNA2核酸酶的乙酰化大大增强了切割活性。乙酰化会刺激DNA聚合酶，从而取代皮瓣。综上所述，这些效应表明了一种规则，即创建更长的瓣片，由DNA2适当处理，但只有在由于FEN1活性较低而更换长补丁后才能连接。这种调控将导致复制/修复蛋白取代较长的补丁，确保在复制过程中去除Okazaki引物，在修复过程中完全消除损伤。这项规定将在忠诚度和效率之间取得平衡。在酵母中有增强的容易出错的DNA聚合酶？的初步证据表明，一种主要蛋白质乙酰酶的缺失降低了DNA复制的保真度，支持这一假说。我们的建议有两个关键部分：第一，复制/修复蛋白的乙酰化引起的酶变化将针对单个蛋白质、相互作用的蛋白质伙伴以及复制和修复途径的重组进行定义。结果应该揭示乙酰化是否改变了与原始假设一致的蛋白质功能，或者提出了替代的解释。第二，将应用遗传学和细胞生物学方法来确定复制/修复蛋白乙酰化在细胞中的影响。我们将具体确定乙酰化是否用于调节复制/修复中的斑块替换长度，以及该调节是否改变DNA合成的保真度。

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.