Structure-Function Studies Of DNA Replication Fidelity

今年，我们发表了关于DNA合成保真度的几个方面的文章。(1)我们完成了两项关于DNA聚合酶lambda合成DNA保真度的研究，Lambda是一种参与修复因暴露于环境应激(包括辐射)而造成的DNA损伤的酶。我们鉴定并鉴定了一个缺失与DNA模板链相互作用的环的突变型聚合酶，并获得了该聚合酶的结构，在活性部位有匹配的或G-T不匹配的碱基对。错配具有Watson-Crick几何结构，并准备用于催化，从而提供了第一个直接的结构证据，支持Watson和Cricks 1953年提出的自发突变的一个起源。(2)我们完成了四项关于rNTPs在复制过程中掺入DNA及其后果的研究。这些后果包括复制压力，以及由于拓扑异构酶1切割链的新机制而导致的基因组不稳定。(3)利用深度测序技术，我们为POL Delta是萌芽酵母整个核基因组的主要滞后链复制酶的观点提供了强有力的支持。(4)我们证明了由核糖核苷酸还原酶突变引起的dNTP池的改变会导致体内的突变模式，这是我们和其他人在过去20年里在体外建立的复制保真度第一原理所预测的。此外，这些模式强烈表明，某些dNTP池不平衡以高度链特异性的方式降低了体内复制的保真度。5)与普遍接受的逻辑相反，我们证明了Pol Zeta可以完全绕过DNA模板中的几个不同的损伤，而不需要其他聚合酶的帮助。6)我们参与了三项合作研究，通过确定(A)幽门螺杆菌DNA聚合酶1，(B)杂交A家族DNA聚合酶，以更有效地扩增古代DNA样本，以及(C)Pols Delta和epsilon复制在哺乳动物基因组中高度丰富的二核苷酸重复序列，来确定DNA合成的保真度。我们还合作研究了DNA聚合酶lambda对碱基切除修复的抑制作用。7)我们还发表了关于DNA复制保真度的三篇综述。

This year, we published on several aspects of DNA synthesis fidelity. (1) We completed two studies on the fidelity of DNA synthesis by DNA polymerase lambda, an enzyme that participates in repairing DNA lesions resulting from exposure to environmental stress, including radiation. We identified and characterized a mutator variant polymerase missing a loop that interacts with the DNA template strand, and we obtained structures of this polymerase with either a matched or a G-T mismatched base pair at the active site. The mismatch has Watson-Crick geometry and is poised for catalysis, thus providing the first direct structural evidence to support Watson and Cricks 1953 proposal for one origin of spontaneous mutations. (2) We completed four studies on the incorporation of rNTPs into DNA during replication and its consequences. These consequences include replicative stress, and genome instability resulting from a novel mechanism involving strand cleavage by topoisomerase 1. (3) Using deep sequencing technology, we provided strong support for the idea that Pol delta is the primary lagging strand replicase for the whole nuclear genome in budding yeast. (4) We demonstrated that alterations in dNTP pools resulting from mutations in ribonucleotide reductase result in patterns of mutagenesis in vivo that are predicted by the first principles of replication fidelity that we and others established in vitro over the past two decades. Moreover, these patterns strongly suggest that certain dNTP pool imbalances reduce replication fidelity in vivo in a highly strand-specific manner. 5) Somewhat contrary to generally accepted logic, we demonstrated that Pol zeta can completely bypassing several different lesions in DNA templates without assistance from other polymerases. 6) We contributed to three collaborative studies by determining the fidelity of DNA synthesis by (a) Helicobacter pylori DNA polymerase 1, (b) by hybrid family A DNA polymerases engineered to more efficiently amplify ancient DNA samples, and (c) by Pols delta and epsilon as they copy dinucleotide repeat sequences that are highly abundant in mammalian genomes. We also collaborated to investigate inhibition of base excision repair catalyzed by DNA polymerase lambda. 7) We also published three reviews on DNA replication fidelity.