Accurate replication of DNA during cellular division is central to maintaining genome integrity where the consequence inaccurate replication is genomic damage leading to cancer and other age related dysfunction. These studies will inform approaches to strengthening mechanism required for accurate DNA replication and predicting the locations where damage is likely to occur when these mechanisms fail. The project is based on a transgenic mouse line developed by this laboratory that carries a subtle mutation resulting in insufficient expression of a core component of the replication machinery, Mcm2, which is proving to be extremely valuable in understanding the consequences of deficient replication origin licensing. These mice are remarkably cancer prone and exhibit complete penetrance of thymic lymphoblastic lymphoma on the 129Sv genetic background on which it was constructed. Studies supported by this project have implicated inefficient initiation of replication from primar origins and, to a greater extent, dormant (backup) origins under conditions of replication fork stalling and collapse as a major reason for this susceptibility. Additionally, the genetic damage in the resulting tumors is in the form of short deletions that markedly facilitate identification o tumor suppressor genes. Further, these studies have shown that some strains of mice differ in their ability to manage the consequences of Mcm2 deficiency as evidenced by reduced tumorigenesis. The proposed studies address three key issues: 1) To what degree does sequence specificity, either directly or as a consequence of epigenetic modification, in the sites at which DNA replication initiates influence the locations at which replication related genetic damage is likely to arise in Mcm2 deficient mice? 2) Do genetic factors that mitigate the disease phenotypes resulting from Mcm2 deficiency on alternative genetic backgrounds function through an effect on the efficiency of origin licensing at specific locations or are there alternative mechanisms that suppress the occurrence or consequences of replication related genetic damage in these strains? 3) To what extent does Mcm2 deficiency reflect changes that occur in cells that are under proliferative stress? Four aims are proposed. The first will define the effect of different genetic backgrounds on the properties and sites of genetic damage that occurs in tumors to address specific mechanisms and genes responsible for tumor suppression. The second will define genome wide the locations at which initiation of replication is most affected by Mcm2 deficiency on different genetic backgrounds. The third will measure the sites at which genetic damage occurs due to Mcm2 deficiency in thymic tissue prior to the formation of tumors to allow correlation between replication efficiency and genetic damage directly, in the absence of any additional selection for tumorigensis. The fourth will determine the extent to which changes observed under conditions of Mcm2 deficiency are also seen under conditions of proliferative stress. These studies will inform approaches to predicting predisposition to genetic damage resulting in specific disease and mitigating the consequences of such predisposition.
This proposal addresses the contribution of DNA damage occurring during cellular replication to cancer initiation and progression. Studies test the hypotheses that sequence specificity in the sites at which DNA replication mechanisms fail lead to specific disease outcomes and that genetic modifiers that reduce susceptibility to replication related genetic damage or its consequences exist and can be identified. These studies will inform approaches to predicting susceptibility to particular genetic damage under conditions of proliferative stress and suppressing the occurrence of this damage or mitigating the disease related consequences.
