Large Loop DNA Repair in Yeast

DNA错配被归类为单碱基错配，如G-T，或一条链上过量核苷酸的环。由于DNA复制错误而出现的错配是诱变前病变。众所周知的错配修复(MR)系统作用于单碱基错对和小环，以防止替换错误和小的插入和缺失。在没有MR的情况下，突变率会大大提高。因此，MR纠正错配的能力提供了一个强大的突变避免系统。具有遗传性MR缺陷的人容易患遗传性非息肉病结肠癌(HNPCC)以及其他类型的癌症。虽然MR对许多错配对有效，但它在大于约13个核苷酸的LOP上不起作用。最近来自酵母和人类细胞的生化证据表明，另一种称为大环修复(LLR)的修复途径在较大的异种上是活跃的。有人认为，LLR通过纠正诱变前的环错对来帮助防止大的插入和缺失。LLR与MR明显不同；例如，LLR在关键MR基因零突变的细胞中完全活跃。这个项目的目标是推断酵母LLR的关键生化方面，识别所需的蛋白质，并开始确定LLR在遗传稳定中的重要性。提出了两种互补的方法，利用本实验室开发的高度特异的生化和遗传分析方法。该项目将有助于通过描述一种新的、保守的、真核生物特异的修复途径来推动DNA修复领域的发展。这项研究还将揭示新的DNA修复蛋白，并提供有关其生化功能的信息。该项目还将解决LLR的遗传影响。之所以选择酵母，是因为在这种有机体中已经证明了环路修复，因为它提供了一个简单的真核系统，而且酵母的实验易操纵性允许人们回答关于环路修复的重要问题。根据对酵母中MR的基础科学方法导致人类HNPCC相关的先例，对酵母LLR的研究也可能有助于确定其他遗传性癌症或遗传病的突变原因。

DNA mismatches are classified as either single base mispairs, like G-T, or loops of excess nucleotides on one strand. Mismatches that arise as errors in DNA replication are pre- mutagenic lesions. The well-known mismatch repair (MR) system acts on single base mispairs and on small loops to prevent substitution errors and small insertions and deletions. In the absence of MR, mutation rates are greatly elevated. The ability of MR to correct mismatches therefore provides a powerful mutation avoidance system. Humans with inherited MR deficiencies are prone to hereditary nonpolyposis colon cancer (HNPCC), as well as other types of cancer. Although MR is effective on many mispairs, it does not function on lops larger than about 13 nt. Recent biochemical evidence from yeast and human cells indicates that another repair pathway called large loop repair (LLR) is active on larger heterologies. It is proposed that LLR helps prevent large insertions and deletions by correcting the pre-mutagenic looped mispair. LLR is clearly distinct from MR; for example, LLR is fully active in cells with null mutations in crucial MR genes. The objectives of this project are to deduce key biochemical aspects of yeast LLR, to identify required proteins, and to begin establishing the importance of LLR in genetic stabilization. Two complementary approaches are proposed that utilize highly specific biochemical and genetic assays developed in this laboratory. This project will help advance the field of DNA repair by characterizing a novel, conserved, eukaryote-specific repair pathway. The research will also reveal new DNA repair proteins and provide information on their biochemical functions. This project will also address the genetic impact of LLR. Yeast was chosen because loop repair has been demonstrated in this organism, because it provides a simple eukaryotic system and because the experimental tractability of yeast allows one to answer important questions about loop repair. Based on the precedent that basic science approaches to MR in yeast led to the connection with human HNPCC, it is suggested that studies of yeast LLR may also help identify the mutational cause of other hereditary cancers or genetic diseases.