DNA damage response and tumorigenesis in Mcm2-deficient mice

Abstract

Mini-chromosome maintenance proteins (Mcm’s) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm proteins 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 MEFs 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 HU mediated replication stress. DNA damage responses (DDR) resulting from HU and additional replication dependent and independent genotoxic agents were also examined and shown to function at wild type levels. Further, basal levels of many components of the DNA damage response were expressed at wild type levels demonstrating that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5–2 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 following UV 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 demonstrate that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. Additionally, these studies demonstrate that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.

Effect of minichromosome maintenance protein 2 deficiency on the locations of DNA replication origins

“…Despite the reduction of dormant origins in MCM2-deficient MEFs (Kunnev et al 2010), the rate of division of these cells is unaffected (Supplemental Fig. S1A-C).…”

“…Locations of reduced nascent strand density correlate with recurrent copy number variations (CNVs) in tumors resulting from MCM2 deficiency MCM2-deficient mice on a 129Sv genetic background develop thymic lymphocytic leukemia (T-LL) with early onset (average age 12 wk) and complete penetrance (Pruitt et al 2007;Kunnev et al 2010). The genetic lesions occurring in these tumors have been characterized and are predominately focal deletions averaging ∼450 kbp in length (Rusiniak et al 2012).…”

“…Under conditions in which MCM protein levels are reduced (Ge et al 2007;Ibarra et al 2008;Kunnev et al 2010), although not necessarily in cases where MCM protein function is affected by mutation (Kawabata et al 2011), it has been shown that inter-ori distance is increased under conditions of replication stress even though the rate of fork progression is not affected. These observations support that replication licensing, rather than the rate of fork movement or stalling, is responsible for the elevated genetic damage and cancers seen in MCM2-deficient mice (Pruitt et al 2007;Kunnev et al 2010).…”

“…The licensing of dormant origins is postulated to serve as a backup system to allow completion of replication in the event of replication fork stalling (Woodward et al 2006;Ge et al 2007;Ibarra et al 2008). The importance of sufficient DNA replication origin licensing to the maintenance of genome stability is demonstrated by the high rates of cancer incidence in mice in which MCM levels or function are reduced (Pruitt et al 2007;Shima et al 2007;Kunnev et al 2010;Kawabata et al 2011;Rusiniak et al 2012). Further, under conditions in which the demand for cell proliferation is high, or where cells are transitioning from a low to an accelerated rate of growth, MCM levels have been shown to be limiting, resulting in a reduced level of primary or dormant origin licensing (Orr et al 2010).…”

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

“…In fission yeast, a reduction in MCM protein levels causes genome instability due to replication fork collapse and DNA damage (10,11). In human cells, excess chromatin-loaded MCM complexes are important under conditions of replicative stress, where they activate dormant origins to ensure that DNA replication continues when the replication forks stall (12,13 Chaos3 ) show lower levels of Mcm2-7 loading onto DNA, exhibit replicative stress even under unchallenged conditions, and have a high incidence of cancer (14,15). In addition, some MCM subunits appear to play additional roles that are independent of DNA replication (16 -18).…”