Abstract
Minichromosome maintenance proteins (Mcm's) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm's has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007) and stem cell deficiencies (Pruitt et al., 2007). Here we use mouse embryonic fibroblasts from an Mcm2-deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2-deficient cells under conditions of hydroxyurea (HU)-mediated replication stress. DNA damage responses (DDRs) resulting from HU and additional replication-dependent and replication-independent genotoxic agents were also examined and shown to function at wild-type (wt) levels. Further, basal levels of many components of the DDR were expressed at wt levels, showing that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5- to 2-fold increases in the basal levels of γ-H2AX, p21cip1 and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt- and Mcm2-deficient cells was found occurred after ultraviolet irradiation and may reflect the role of Chk1-mediated suppression of dormant origins. In vivo, abrogating p53-mediated DDR in Mcm2-deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results show that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. In addition, these studies show that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.
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Acknowledgements
BALB/c:p53null/wt mice were a generous gift of Dr Peter Demant. This work was supported by grants from the NIH-NCI, the Ellison Medical Foundation, and NYSTEM to SCP. Cost of animal maintenance and flow cytometry was supported in part by an NCI-CCS grant to RPCI.
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Dr Pruitt's work has been funded by the NIH. He is the founder and owner of the Buffalo Molecular Target Laboratories. Dr Kunnev, Dr Rusiniak, Ms Kudla, Ms Freeland and Ms Cady declare no potential conflict of interest.
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Kunnev, D., Rusiniak, M., Kudla, A. et al. DNA damage response and tumorigenesis in Mcm2-deficient mice. Oncogene 29, 3630–3638 (2010). https://doi.org/10.1038/onc.2010.125
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DOI: https://doi.org/10.1038/onc.2010.125