Assessment of DNA fibers to track replication dynamics.

DNA replication is a complex and tightly regulated process that must proceed accurately and completely if the cell is to faithfully transmit genetic material to its progeny. Organisms have thus evolved complex mechanisms to deal with the myriad exogenous and endogenous sources of replication impediments to which the cell is subject. These mechanisms are of particular relevance to cancer biology, given that such "replication stress" frequently foreshadows genome instability during cancer pathogenesis, and that many traditional chemotherapies and a number of precision medicines function by interfering with the progress of DNA replication. Visualization of the progress and dynamics of DNA replication in living cells was historically a major challenge, neatly surmounted by the development of DNA fiber assays that utilize the fluorescent detection of halogenated nucleotides to track replication forks at single-molecule resolution. This methodology has been widely applied to study the dynamics of unperturbed DNA replication, as well as the cellular responses to various replication stress scenarios. In recent years, subtle modifications to DNA fiber assays have facilitated assessment of the stability of nascent DNA at stalled replication forks, as well as the detection of single-stranded DNA gaps and their subsequent filling by error-prone polymerases. Here, we present and discuss several iterations of the fiber assay and suggest methodologies for the analysis of the data obtained.

MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.