No abstract

ABSTRACThomozygous embryos or mouse embryonic fibroblasts, Mcm2 is reduced to approximately one-third of wild-type levels. Despite the fact that these mice develop normally and are asymptomatic as young adults, life span is greatly reduced, with most surviving to only ϳ10 -12 weeks of age. They demonstrate severe deficiencies in the proliferative cell compartments of a variety of tissues, including the subventricular zone of the brain, muscle, and intestinal crypts. However, the immediate cause of death in most of these animals is cancer, where the majority develop lymphomas. These studies directly demonstrate that deficiencies in the function of the core DNA replication machinery that are compatible with development and survival nonetheless result in a chronic phenotype leading to stem cell deficiency in multiple tissues and cancer. STEM CELLS 2007;25:3121-3132 Disclosure of potential conflicts of interest is found at the end of this article.

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.

From 1990 to 2004, the reported rates of diarrheal disease (three or more loose stools or a greater than normal frequency in a 24-hour period) on cruise ships decreased 2.4%, from 29.2 cases per 100,000 travel days to 28.5 cases (1,2). Increased rates of acute gastroenteritis illness (diarrhea or vomiting that is associated with loose stools, bloody stools, abdominal cramps, headache, muscle aches, or fever) occurred in years that novel strains of norovirus, the most common etiologic agent in cruise ship outbreaks, emerged (3). To determine recent rates of acute gastroenteritis on cruise ships, CDC analyzed combined data for the period 2008-2014 that were submitted by cruise ships sailing in U.S. jurisdiction (defined as passenger vessels carrying ≥13 passengers and within 15 days of arriving in the United States) (4). CDC also reviewed laboratory data to ascertain the causes of acute gastroenteritis outbreaks and examined trends over time. During the study period, the rates of acute gastroenteritis per 100,000 travel days decreased among passengers from 27.2 cases in 2008 to 22.3 in 2014. Rates for crew members remained essentially unchanged (21.3 cases in 2008 and 21.6 in 2014). However, the rate of acute gastroenteritis was significantly higher in 2012 than in 2011 or 2013 for both passengers and crew members, likely related to the emergence of a novel strain of norovirus, GII.4 Sydney (5). During 2008-2014, a total of 133 cruise ship acute gastroenteritis outbreaks were reported, 95 (71%) of which had specimens available for testing. Among these, 92 (97%) were caused by norovirus, and among 80 norovirus specimens for which a genotype was identified, 59 (73.8%) were GII.4 strains. Cruise ship travelers experiencing diarrhea or vomiting should report to the ship medical center promptly so that symptoms can be assessed, proper treatment provided, and control measures implemented.

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number.