Methods for the detection of genome instability derived from replication stress in primary mouse embryonic fibroblasts.

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.

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression.

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.

Helq acts in parallel to Fancc to suppress replication-associated genome instability.

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.

A reduction of licensed origins reveals strain-specific replication dynamics in mice.

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.

Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression.

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 Mcm4(Chaos3) allele.