Summary 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.

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive. Here, we investigated this question in mice using the Helqgt and Fancc− strains. Compared with Fancc−/− mice lacking FANCC, a component of the FA core complex, Helqgt/gt mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc−/− primary fibroblasts, Helqgt/gt cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helqgt/gt;Fancc−/− double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.

Replication origin licensing builds a fundamental basis for DNA replication in all eukaryotes. This occurs during the late M to early G1 phases in which chromatin is licensed by loading of the MCM2-7 complex, an essential component of the replicative helicase. In the following S phase, only a minor fraction of chromatin-bound MCM2-7 complexes are activated to unwind the DNA. Therefore, it is proposed that the vast majority of MCM2-7 complexes license dormant origins that can be used as backups. Consistent with this idea, it has been repeatedly demonstrated that a reduction (~60%) in chromatin-bound MCM2-7 complexes has little effect on the density of active origins. In this study, however, we describe the first exception to this observation. A reduction of licensed origins due to Mcm4chaos3 homozygosity reduces active origin density in primary embryonic fibroblasts (MEFs) in a C57BL/6J (B6) background. We found that this is associated with an intrinsically lower level of active origins in this background compared to others. B6 Mcm4chaos3/chaos3 cells proliferate slowly due to p53-dependent upregulation of p21. In fact, the development of B6 Mcm4chaos3/chaos3 mice is impaired and a significant fraction of them die at birth. While inactivation of p53 restores proliferation in B6 Mcm4chaos3/chaos3 MEFs, it paradoxically does not rescue animal lethality. These findings indicate that a reduction of licensed origins may cause a more profound effect on cell types with lower densities of active origins. Moreover, p53 is required for the development of mice that suffer from intrinsic replication stress.

Accumulating evidence suggests that dormant DNA replication origins play an important role in the recovery of stalled forks. However, their functional interactions with other fork recovery mechanisms have not been tested. We previously reported intrinsic activation of the Fanconi anemia (FA) pathway in a tumor-prone mouse model (Mcm4chaos3) with a 60% loss of dormant origins. To understand this further, we introduced a null allele of Fancc (Fancc−), encoding a member of the FA core complex, into the Mcm4chaos3 background. Primary embryonic fibroblasts double homozygous for Mcm4chaos3 and Fancc− (Mcm4chaos3/chaos3;Fancc−/−) showed significantly increased levels of markers of stalled/collapsed forks compared to either single homozygote. Interestingly, a loss of dormant origins also increased the number of sites in which replication was delayed until prophase, regardless of FA pathway activation. These replication defects coincided with substantially elevated levels of genome instability in Mcm4chaos3/chaos3;Fancc−/− cells, resulting in a high rate of perinatal lethality of Mcm4chaos3/chaos3;Fancc−/− mice and the accelerated tumorigenesis of surviving mice. Together, these findings uncover a specialized role of dormant origins in replication completion while also identifying important functional overlaps between dormant origins and the FA pathway in maintaining fork progression, genome stability, normal development and tumor suppression.

Replication stress, with its subsequent genome instability, is a hallmark of cancer from its earliest stages of development. Here, we describe assays that are sufficiently sensitive to detect intrinsic replicative stress and its consequences in primary mouse embryonic fibroblasts. First, we explain the non-denatured DNA fiber assay, a powerful tool to directly measure DNA replication kinetics via the dual-labeling of active replication forks. Then, we describe the cytokinesis-block micronucleus assay, which can be combined with detection of 53BP1 nuclear bodies to measure the levels of replication-associated genome instability carried over into G1 phase of the cell cycle.