This proposal addresses basic mechanisms by which DNA replication origin function maintains genome stability and the contribution of deficient replication origin licensing to the genomic changes that drive cancer. During origin licensing, origin recognition complex (ORC), Cdc6 and Cdt1 load an inactive form of the minichromosome maintenance protein (MCM) 2-7 hexameric helicase onto chromatin to form pre-replication complexes (pre-RC). At S-phase the helicase activity of MCM2-7 is activated to initiate replication. MCM2-7 is loaded onto chromatin in excess where the excess complexes are dormant unless required to reinitiate replication in the event of stalling of adjacent active replication forks. Using a transgenic mouse model in which MCM2 levels are reduced to ~1/3 of normal, we have shown that insufficient DNA replication origin licensing is a potent driver of replication related errors, predominately focal deletions, leading to loss of tumor suppressor genes and cancer. Using a novel method of short nascent strand analysis to map replication origins we have shown that limiting concentrations of MCM2-7 leads to preferential loss of licensing at discrete genomic locations. A subset of these locations correlates with sites of recurrent focal deletions in tumors that arise in these mice and affect tumor suppressor genes. Further, genetic background affects locations where origin function is lost in parallel with the locations where focal deletions occur and correlate with tumor incidence in MCM2 deficient mice. Here we will directly test the relationship between replication origin function, genomic damage, and cancer incidence by disrupting an origin adjacent to a tumor suppressor gene. Specifically, in aim 1 we will refine a methodology for measuring under-replicated regions of the genome and characterize these regions genome wide in wt and MCM2 deficient mice.
In aim 2 we will utilize this method as a readout for under-replication to assay a systematic deletion series across a 60 kbp domain in wt and MCM2 deficient MEFs and map sequences required for origin function. The relationship between functional elements and sites identified by SNS analysis as active origins will be established. The 60 kbp domain contains a tumor suppressor gene and once elements required for origin function are defined, we will target these elements using CRISPR-Cas9 to generate a mouse model in which origin function at this location is lost. These mice will be crossed with MCM2 deficient mice and the consequences of loss of origin function on deletion of the tumor suppressor gene and tumor incidence will be determined in wt mice in comparison to MCM2 deficient mice in which the ability of cells to compensate for loss of the origin by activation of dormant origins is limited.
In aim 3 we will examine the interaction between loss of origin function and replication stress induced by chemotherapeutic agents (HU and gemcitabine) in determining sites of chromosome fragility. The effects of modulating MCM2-7 levels using CDK inhibitors on HU and gemcitabine sensitive sites will be determined.

Public Health Relevance

The larger objective of the present studies is to improve our understanding of mechanisms underlying genome instability in the etiology of cancer and the effects of genetic background on this instability. A unique set of genetically engineered mouse models and methods will be used to examine the role of replication licensing in maintaining genome stability and the consequences of deficiency in replication licensing factors for genome locations susceptible to damage. Additionally, this work will inform the role of replication licensing status in normal tissues and cancer on locations at which genotoxic agents induce genetic damage.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA130995-11
Application #
9522485
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Pelroy, Richard
Project Start
2008-04-06
Project End
2023-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
11
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
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