DNA REPLICATION AND GENOME STABILITY

描述（由申请方提供）：本研究的长期目标是了解负责从染色体DNA插入和去除核糖核苷单磷酸（rNMP）的机制。遗传物质的精确复制对所有活细胞都是必不可少的。最近，DNA聚合酶直接将核糖糖作为rNMP掺入DNA中已经变得明显。DNA的一个特点是它是化学稳定的，比RNA反应性低得多。核糖上的2'羟基使rNMP的反应性增加100，000倍，导致在正常生理条件下水解和DNA断裂。此外，rNTPs的细胞内浓度远远超过dNTPs的浓度，导致它们在复制过程中错误插入染色体DNA。rNMP掺入的错误率表明，每约103个正确配对的碱基发生错误掺入，使得rNMP错误远远超过体内任何类型的复制错误或受损碱基。我们发现细菌中的复制型DNA聚合酶经常将rNMP掺入DNA中。在这项工作中，我们将阐明插入，删除的机制，以及当rNMP未修复时对基因组完整性的影响。此外，我们发现了一种新的蛋白质，将rNMP去除与基因组完整性联系起来，为rNMP错误提供了进化益处。整合的rNMP对人类健康有深远的影响。核糖核苷单磷酸减慢DNA合成，rNMP具有致突变潜力。此外，负责从DNA中去除单个rNMP的RNA酶H2在小鼠中是必需的，并且人RNA酶H2的突变导致称为Aicardi-Goutieres综合征的神经系统疾病。因此，我们对rNMP插入和去除的研究对人类健康具有实际意义。我们的具体目标是：1）确定rNMP体外掺入率; 2）确定rNMP去除机制; 3）确定rNMP去除对基因组完整性的进化益处。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the mechanisms responsible for insertion and removal of ribonucleoside monophosphates (rNMPs) from chromosomal DNA. The accurate duplication of genetic material is essential for all living cells. Recently, it has become apparent that DNA polymerases directly incorporate ribose sugars into DNA as rNMPs. A hallmark of DNA is that it is chemically stable and much less reactive than RNA. The 2' hydroxyl on the ribose sugar causes rNMPs to be 100,000 fold more reactive resulting in hydrolysis and DNA breaks under normal physiological conditions. Furthermore, the intracellular concentration of rNTPs far exceeds that of dNTPs contributing to their misinsertion into chromosomal DNA during replication. Error rates for rNMP incorporation suggest misincorporation occurs every ~103 correctly paired bases making rNMP errors far exceed that of any type of replication error or damaged base in vivo. We have found that the replicative DNA polymerases in bacteria frequently incorporation rNMPs into DNA. In this work, we will elucidate the mechanisms of insertion, removal, and the consequences to genome integrity when rNMPs are left unrepaired. Moreover, we have found a novel protein that links rNMP removal to genome integrity providing an evolutionary benefit for rNMP errors. Incorporated rNMPs have profound effects on human health. Ribonucleoside monophosphates slow DNA synthesis and rNMPs have mutagenic potential. Furthermore, RNase H2 the enzyme responsible for removing single rNMPs from DNA is essential in mice and mutations in human RNase H2 results in a neurological disorder known as Aicardi-Goutieres syndrome. Thus, our studies of rNMP insertion and removal have practical implication for human health. Our specific aims are: 1) to determine the rate of rNMP incorporation in vitro; 2) determine the mechanisms of rNMP removal; 3) determine the evolutionary benefit of rNMP removal to genome integrity.