DNA Mismatch Repair Functions in Tumor Suppression

描述（由申请人提供）：DNA错配修复（MMR）基因的杂合性种系缺陷导致遗传性非息肉病性结直肠癌（HNPCC），这是最常见的遗传性癌症综合征之一，而纯合性缺陷导致严重的血液系统恶性肿瘤。在其他散发性癌症中也发现了MMR缺陷，这表明MMR缺失是人类肿瘤发生的常见病因。在细胞水平上，MMR通过纠正DNA复制过程中的错误和识别导致生长停滞和细胞死亡的几种类型的DNA损伤来确保基因组的稳定性。这些不同功能的确切机制尚不清楚。特别不明确的是对DNA损伤的核磁共振依赖反应。缺乏MMR的细胞表现出更高的自发突变率和对DNA损伤剂诱导的细胞杀伤的抵抗力。然而，与MMR缺乏相关的不同表型在多大程度上导致癌症风险尚不清楚。为了阐明MMR在预防癌症中的作用机制和重要性，我们提出了MMR基因MLH1的功能表征。我们将通过分析表达MLH1变异的等基因细胞系来确定MLH1特定突变的表型后果（目的1）。我们将通过错配定向切除、错误纠正和DNA损伤处理的生化分析来阐明在目标1中发现的遗传缺陷的机制（目标2）。最后，我们将使用一种独特的遗传选择来识别和表征MMR基因中的新突变，这种突变会干扰MMR对DNA损伤的依赖反应（目的3）。我们的分析将增加对MMR机制的理解，特定MMR基因突变的后果，以及不同MMR功能对预防癌症的重要性。这些知识将有助于更合理地设计干预策略，以治疗和/或预防与MMR缺乏相关的癌症。

DESCRIPTION (provided by applicant): Heterozygous germline deficiencies in DNA mismatch repair (MMR) genes cause hereditary non-polyposis colorectal cancer (HNPCC), one of the most common inherited cancer syndromes, whereas homozygous deficiency causes severe hematological malignancy. Defects in MMR also are detected in other sporadic cancers, suggesting that loss of MMR is a common etiologic factor in human tumorigenesis. At the cellular level, MMR ensures the stability of the genome through the correction of errors made during DNA replication and the recognition of several types of DNA damage, leading to growth arrest and cell death. The precise mechanism of these different functions is unknown. Particularly poorly defined are the MMR-dependent responses to DNA damage. Cells lacking MMR demonstrate an elevated rate of spontaneous mutation and resistance to cell killing induced by DNA damaging agents. However, the extent to which the different phenotypes associated with MMR deficiency contribute to cancer risk is not known. To elucidate the mechanisms and importance of MMR functions in the prevention of cancer, we propose a functional characterization of the MMR gene MLH1. We will define the phenotypic consequences of specific mutations in MLH1 through the analysis of isogenic cell lines expressing MLH1 variants (Aim 1). We will elucidate the mechanism underlying genetic deficiencies identified in Aim 1 using biochemical assays of mismatch-directed excision, error correction, and DNA damage processing (Aim 2). Finally, we will identify and characterize novel mutations in MMR genes using a unique genetic selection for dominant-acting mutations that interfere with MMR-dependent responses to DNA damage (Aim 3). Our analyses will increase understanding of the mechanisms of MMR, the consequences of specific MMR gene mutations, and the importance of different MMR functions for the prevention of cancer. Such knowledge will facilitate more rationally designed intervention strategies for the treatment and/or prevention of cancer associated with MMR deficiency.