Unraveling the molecular mechanisms of DNA-protein crosslink repair in mammals and understanding its impact on human health

National Institute Of Environmental Health Sciences

Investigators

Abstract

Deleterious single-strand DNA nicks routinely arise during mammalian DNA replication. Replication fork collision with a single-strand DNA nick can generate a one-ended break, fostering genomic instability. It is speculated that the opposing replication fork can collide with a DNA nick generating a second free DNA end, enabling conventional and error-free repair by the homologous recombination (HR) pathway. To study mechanisms of nickase-induced HR in mammalian cells, we developed a site-specific Flp recombinase “step arrest” nickase reporter system. In this system, the Flp recombinase generates a DNA nick and stays covalently bound to the 3’ DNA nick, similar to a lesion generated by the chemotherapeutic drug camptothecin. We noted that a Flp-nick induces two-ended conservative, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and a unusual discoordinated two-ended invasions. Interestingly, HR pathways induced by a replication-independent break differ from the Flp-nicks in their dependence on key HR proteins BRCA1, MRE11, and CtIP. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using another site-specific replication fork barrier. Flp-nickase-induced STGC remained robust and two ended suggesting a single replication fork’s collision with a Flp-nick triggers two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. Our results unravel a novel mechanism in which cells can limit genomic instability during replication of nicked DNA in mammalian cells. In summary, we find that a major product of DNA-protein crosslink-nick-induced fork breakage is two-ended, produces error-free STGC events and that this pathway requires the arrival of only one replication fork at the nick site. This error-free pathway may serve as a barrier against genomic instability during replication of a nicked DNA template. This allows us to understand the mechanism by which clinical DNA nickases can have valuable therapeutic implications. Specifically, gene-editing nickases that target the parental lagging strand may carry a lower risk of genomic instability than those that target the parental leading strand. It is critical to test this hypothesis as these new therapeutic tools progress toward use in the clinic.