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.
|Pruitt, Steven C; Qin, Maochun; Wang, Jianmin et al. (2017) A Signature of Genomic Instability Resulting from Deficient Replication Licensing. PLoS Genet 13:e1006547|
|Salim, Devika; Bradford, William D; Freeland, Amy et al. (2017) DNA replication stress restricts ribosomal DNA copy number. PLoS Genet 13:e1007006|
|Kunnev, Dimiter; Freeland, Amy; Qin, Maochun et al. (2015) Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR). J Biol Methods 2:|
|Kunnev, Dimiter; Freeland, Amy; Qin, Maochun et al. (2015) Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins. Genome Res 25:558-69|
|Morrison, Carl D; Liu, Pengyuan; Woloszynska-Read, Anna et al. (2014) Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer. Proc Natl Acad Sci U S A 111:E672-81|
|Pruitt, Steven C; Freeland, Amy; Rusiniak, Michael E et al. (2013) Cdkn1b overexpression in adult mice alters the balance between genome and tissue ageing. Nat Commun 4:2626|
|Rusiniak, M E; Kunnev, D; Freeland, A et al. (2012) Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma. Oncogene 31:4034-44|
|Chuang, Chen-Hua; Yang, Dian; Bai, Gongshi et al. (2012) Post-transcriptional homeostasis and regulation of MCM2-7 in mammalian cells. Nucleic Acids Res 40:4914-24|
|Kunnev, D; Rusiniak, M E; Kudla, A et al. (2010) DNA damage response and tumorigenesis in Mcm2-deficient mice. Oncogene 29:3630-8|
|Pruitt, Steven C; Freeland, Amy; Kudla, Angela (2010) Cell cycle heterogeneity in the small intestinal crypt and maintenance of genome integrity. Stem Cells 28:1250-9|