DNA Replication Fidelity and Cancer

描述（由申请人提供）：正常细胞高保真复制其基因组（约10-10个突变/nt/复制周期）。这是通过聚合酶碱基选择性、核酸外切校正（exo）、错配校正和DNA损伤修复的组合作用来实现的。我们最近创建了在DNA聚合酶d（Pol d）的3 '->5'外切核酸水解校对结构域中具有失活点突变（Pold 1D 400 A）的“敲入”小鼠，并表明纯合突变体产生了独特的早发性上皮肿瘤谱（皮肤和肺，但不是肠）。在这里，我们建议使用这些新的Pol delta外小鼠和衍生细胞系来表征Pol delta校正在维持哺乳动物遗传稳定性和避免癌症中的作用。具体目标是： 1.表征Pol δ外细胞和小鼠的增变子表型。为了确定由Pol delta校正丢失所赋予的增变基因表型的程度和性质，我们将确定培养物和体内组织中Poldelta 1D 400 A细胞中产生的自发突变的速率、频率和光谱。 2.检查Pol delta校对与错配修复（MMR）的协同性。将研究携带Pol delta校正和MMR等位基因组合的小鼠和细胞，以评估这些纠错系统如何在哺乳动物中在表型水平（存活、突变和癌症表型）上合作。 总之，这些研究将大大有助于我们理解癌症中的增变因子表型，并将表征Pol delta校正对哺乳动物突变和癌症避免的贡献。

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.