Replication Licensing in Genome Stability, Cancer and Aging

描述（由申请人提供）：长期以来，基因组不稳定性被认为在癌症病因学和年龄相关功能障碍中起作用。对负责介导DNA损伤后细胞周期停滞和修复受损DNA的基因突变表型的研究表明，共同的机制影响这两个过程。导致损伤的机制尚未得到很好的定义，可能有多种原因;然而，在严重依赖不断分裂的干细胞的生物体中，如哺乳动物，突变的可能来源是DNA复制。DNA的复制是通过调节DNA复制起点的使用来严格控制的。为了防止DNA的核内复制，复制起点是特异性的，并用于细胞周期的离散阶段。在G1期早期，一种称为许可因子的蛋白质复合物的结合指定了可用于复制的位点。在G1中的一个点，称为限制点，在S阶段完成之前，防止许可因子的额外结合。通过将Cre重组酶的他莫昔芬依赖性形式靶向整合到Mcm 2基因中，该基因是包含许可因子复合物的蛋白质之一，我们偶然地产生了该基因的等位基因，其在小鼠中的表达是亚型的。此外，初步研究表明，该等位基因纯合的小鼠具有高度升高的癌症发病率。此外，它们严重缺乏大脑SVZ内的神经干/祖细胞和骨骼肌内的卫星细胞。本研究旨在确定Mcm 2表达缺陷导致这些表型的机制。待检验的一个假设是亚纯型Mcm 2表达导致较高的复制错误率。指定复制起点所需的确切序列是松散定义的，部分取决于许可因素的浓度。因此，细胞中许可因子的浓度可能会影响被利用的来源数量和DNA复制的效率，从而导致遗传损伤。或者，有证据表明，Mcm蛋白，包括Mcm 2，也在基因表达的控制和在S期的DNA损伤反应的早期阶段中起作用。旨在测试这些潜在的机制是负责升高的癌症和干细胞缺乏表型的目的提出。定义负责这种极端表型的机制将产生重要的见解癌症和年龄相关的功能障碍的病因。长期以来，基因组不稳定性被认为在癌症的病因学和年龄相关的功能障碍中起作用。本研究旨在确定控制DNA复制的蛋白质之一Mcm 2的缺陷导致这些表型的机制。定义这种机制将产生重要的见解癌症和年龄相关的功能障碍的病因。

DESCRIPTION (provided by applicant): Genomic instability has long been thought to play a role in both the etiology of cancer and in age related dysfunction. Studies of the phenotypes of mutations in the genes responsible for mediating cell cycle arrest following DNA damage and repairing damaged DNA suggest that common mechanisms affect both processes. The mechanisms leading to damage have been less well defined and may have multiple causes; however, in organisms such as mammals that depend heavily on continuously dividing stem cells a likely source of mutations is DNA replication. Replication of DNA is tightly controlled through the regulation of DNA replication origin usage. To prevent endoreduplication of the DNA, replication origins are specific and used in discrete phases of the cell cycle. During early G1-phase, the binding of a complex of proteins termed licensing factors specifies the sites that can be utilized for replication. At a point in G1, termed the restriction point, additional binding of licensing factors is prevented until S-phase is complete. Through the targeted integration of a tamoxifen dependent version of Cre-recombinase into the Mcm2 gene, which is one of the proteins comprising the licensing factor complex, we have fortuitously created an allele of this gene which is hypomorphic in its expression in mice. Further, preliminary studies demonstrate that mice which are homozygous for this allele have a highly elevated rate of cancer. Additionally, they are severely deficient in neural stem/progenitor cells within the SVZ of the brain and satellite cells within the skeletal muscle. The present study seeks to define the mechanism by which deficiency in Mcm2 expression results in these phenotypes. One hypothesis to be tested is that hypomorphic Mcm2 expression results in a higher rate of replication errors. The exact sequences required for specifying replication origins are loosely defined and, in part, depend on the concentration of licensing factors. Hence the concentration of licensing factors in the cell may affect the number of origins that are utilized and the efficiency with which DNA is replicated leading to genetic damage. Alternatively, there is evidence that Mcm proteins, including Mcm2, also function in both the control of gene expression and in an early phase of the DNA damage response during S-phase. Aims designed to test which of these potential mechanisms is responsible for the elevated cancer and stem cell deficiency phenotypes are proposed. Defining the mechanism responsible for this extreme phenotype will yield important insights into the etiology of cancer and age related dysfunction. Genomic instability has long been thought to play a role in both the etiology of cancer and in age related dysfunction. The present study seeks to define the mechanism by which deficiency in one of the proteins controlling DNA replication, Mcm2, results in these phenotypes. Defining this mechanism will yield important insights into the etiology of cancer and age related dysfunction.