Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4Chaos3 allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.
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We thank Kazuto Sugimura for the DNA fiber protocol and Bevin Engelward for the FYDR mice, as well as David Largaespada, Anja Bielinsky, and Alexandra Sobeck for their critical reading of the manuscript. We also thank LeAnn Oseth and Dr. B. Hirsch for the G-banding analyses and interpretations, as well as Andy Lane for his assistance in the live-cell imaging experiments. This study was supported by grants (to N.S.) from Susan G. Komen for the Cure (BCTR0707864) and the NCI (R01CA148806). The work at UC Berkeley was supported by the NIH grants CA R37-30490 (to M.R.B.).