Large Loop DNA Repair in Yeast

DNA错配分为单碱基错配，如G-T，或单链上过量核苷酸环。由DNA复制错误引起的错配是致突变前病变。众所周知的错配修复（MR）系统作用于单碱基错配和小环，以防止替代错误和小插入和删除。在没有MR的情况下，突变率大大提高。因此，磁共振校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性结肠癌（HNPCC），以及其他类型的癌症。尽管MR对许多错配都有效，但它对大于13nt的环不起作用。最近来自酵母和人类细胞的生化证据表明，另一种被称为大环修复（LLR）的修复途径对更大的异质有活性。有人提出，LLR通过纠正诱变前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基因无突变的细胞中完全活跃。该项目的目标是推断酵母LLR的关键生化方面，确定所需的蛋白质，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，利用高度特异性的生化和遗传分析在这个实验室开发。该项目将有助于推进DNA修复领域的特征，一个新的，保守的，真核细胞特异性修复途径。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。该项目还将研究LLR的遗传影响。之所以选择酵母，是因为在这种生物体中已经证明了环修复，因为它提供了一个简单的真核系统，因为酵母的实验可追溯性允许人们回答有关环修复的重要问题。基于酵母中MR的基础科学方法导致与人类HNPCC的联系的先例，我们建议酵母LLR的研究也可能有助于确定其他遗传性癌症或遗传性疾病的突变原因。

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.