DNA Replication Fidelity and Cancer

描述(申请人提供)：正常细胞高保真地复制它们的基因组(大约10-10个突变/nT/复制周期)。这是通过聚合酶碱基选择性、核酸外切校对(Exo)、错配校正和DNA损伤修复的综合作用实现的。我们最近在DNA聚合酶d(Pold)的3‘--&gt；5’外切核解核校正域(Pold)中创造了具有失活点突变(Pold1D400A)的“敲入”小鼠，并表明纯合子突变会发展出一种独特的早发性上皮性肿瘤(皮肤和肺，但不是肠道)谱系。在这里，我们建议使用这些新的Pol Delta外鼠及其衍生细胞系来表征Pol Delta校对在维持哺乳动物遗传稳定性和避免癌症方面的作用。具体目标是： 1.鉴定Pol Delta外源细胞和小鼠的突变子表型。为了确定由于失去Pol Delta校对而导致的突变子表型的程度和性质，我们将测定Poldelta1D400A细胞在培养和体内组织中产生的自发突变的比率、频率和谱。 2.检验Pol Delta校对与错配修复(MMR)的协同性。将对携带Pol Delta校对和MMR等位基因组合的小鼠和细胞进行研究，以评估这些纠错系统如何在哺乳动物的表型水平(存活表型、突变表型和癌症表型)上合作。 总之，这些研究将大大有助于我们了解癌症中的突变子表型，并将表征Pol Delta校对对哺乳动物避免突变和癌症的贡献。

DESCRIPTION (provided by applicant): Normal cells replicate their genomes with high fidelity (approximately 10-10 mutations/nt/replication cycle). This is achieved through the combined actions of polymerase base selectivity, exonucleolytic proofreading (exo), mismatch correction and DNA damage repair. We recently created "knock-in" mice with an inactivating point mutation (Pold1D400A) in the 3'-->5' exonucleolytic proofreading domain of DNA polymerase d (Pol d) and showed that homozygous mutants develop a unique spectrum of early-onset epithelial tumors (skin and lung, but not intestine). Here we propose to use these novel Pol delta exo-mice and derivative cell lines to characterize the role of Pol delta proofreading in the maintenance of genetic stability and avoidance of cancer in mammals. The specific aims are: 1. Characterize the mutator phenotype of Pol delta exo-cells and mice. To ascertain the extent and nature of the mutator phenotype conferred by loss of Pol delta proofreading, we will determine rates, frequencies and spectra of spontaneous mutations arising in Poldelta1D400A cells in culture and tissues in vivo. 2. Examine cooperativity of Pol delta proofreading with mismatch repair (MMR). Mice and cells carrying combinations of Pol delta proofreading and MMR alleles will be studied to assess how these error correction systems cooperate in mammals at the phenotypic level (survival, mutator and cancer phenotypes). Together, these studies will significantly contribute to our understanding of mutator phenotypes in cancer and will characterize the contribution of Pol delta proofreading to mutation- and cancer-avoidance in mammals.