Determining the Function of HMCES in DNA Replication and Repair

项目总结/摘要 准确和及时的DNA复制对于保持基因组完整性至关重要。复制叉 经常遇到的障碍，如DNA损伤所造成的环境和内源性遗传毒性 与转录机制的冲突，以及阻碍复制DNA的不寻常的DNA结构 聚合酶导致叉停滞。有大量的蛋白质，有助于解决停滞的 叉子最近，我们的实验室鉴定了5-羟甲基胞嘧啶结合ES细胞特异性（HMCES），或C3 orf 37，作为一种免疫调节剂。 在复制叉处富集的蛋白质。HMCES含有一个SOS反应相关蛋白（SRAP）结构域 在进化上是通过细菌保守的。一项比较基因组学研究发现， HMCES基因与细菌中的SOS操纵子关系密切，提示其具有响应DNA损伤的功能。 然而，迄今为止，还没有任何HMCES的功能研究。我们的初步数据显示， HMCES缺陷的人类细胞具有缓慢的复制动力学，对遗传毒性剂的敏感性增加，并且 增变基因表型HMCES被募集到复制叉和UV损伤的染色质。HMCES结合DNA 并直接与复制体的组分相互作用。确定HMCES在基因组中的功能 维护，我将定义HMCES如何结合DNA和HMCES定位到复制叉和UV 使用诱变、生物化学和结构方法破坏染色质。我会用遗传学的方法 以测试HMCES是否直接作用于复制叉以促进DNA合成的准确完成。

Project Summary/Abstract Accurate and timely DNA replication is critical for maintaining genomic integrity. Replication forks frequently encounter obstacles such as DNA lesions caused by environmental and endogenous genotoxic agents, conflicts with transcriptional machinery, and unusual DNA structures that block the replicative DNA polymerases leading to fork stalling. There are a large number of proteins that facilitate the resolution of stalled forks. Recently, our lab identified 5-hydroxymethylcytosine binding ES cell-specific (HMCES), or C3orf37, as a protein enriched at replication forks. HMCES contains one SOS Response Associated Protein (SRAP) domain that is evolutionarily conserved through bacteria. A comparative genomics study found that the bacterial HMCES gene is close to SOS operons in bacteria suggesting a function in responding to DNA damage. However, to date, there have not been any functional studies of HMCES. Our preliminary data shows that HMCES-deficient human cells have slow replication kinetics, increased sensitivity to genotoxic agents, and a mutator phenotype. HMCES is recruited to replication forks and UV-damaged chromatin. HMCES binds DNA and interacts directly with components of the replisome. To determine the function of HMCES in genome maintenance, I will define how HMCES binds DNA and HMCES localization to replication forks and UV damaged chromatin using mutagenesis, biochemical, and structural approaches. I will use genetic approaches to test whether HMCES directly acts at replication forks to facilitate accurate completion of DNA synthesis.