DNA REPLICATION AND GENOME STABILITY

描述(申请人提供)：这项研究的长期目标是了解负责插入和去除染色体DNA中的核糖核苷一磷酸(RNMP)的机制。遗传物质的准确复制对所有活细胞来说都是必不可少的。近年来，DNA聚合酶以rNMPs的形式直接将核糖结合到DNA中。DNA的一个特点是它在化学上是稳定的，而且比RNA活性小得多。在正常生理条件下，核糖链上的2‘-羟基导致rNMPs的活性增加100,000倍，从而导致水解和DNA断裂。此外，rNTPs的细胞内浓度远远超过dNTPs，导致它们在复制过程中错误插入到染色体DNA中。RNMP掺入的错误率表明，每103个正确配对的碱基就会发生误掺，使rNMP的错误远远超过体内任何类型的复制错误或碱基受损。我们发现细菌中的复制DNA聚合酶经常将rNMPs掺入DNA中。在这项工作中，我们将阐明插入、移除的机制，以及当rNMP不修复时对基因组完整性的影响。此外，我们还发现了一种新的蛋白质，它将rNMP的去除与基因组完整性联系起来，为rNMP错误提供了进化上的好处。被掺入的rNMP对人类健康有着深远的影响。核糖核苷一磷酸使DNA合成缓慢，rNMPs具有诱变潜力。此外，负责从DNA中移除单个rNMPs的RNase H2对小鼠是必不可少的，而人类RNase H2的突变会导致一种被称为Aicardi-Goutieres综合征的神经疾病。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)测定rNMP的体外掺入速率；2)确定rNMP去除的机制；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.