One-ended and two-ended breaks at nickase-broken replication forks

Abstract

Replisome collision with a nicked parental DNA template can lead to the formation of a replication-associated double strand break (DSB). How this break is repaired has implications for cancer initiation, cancer therapy and therapeutic gene editing. Recent work shows that collision of a replisome with a nicked DNA template can give rise to either a single-ended (se) or a double-ended (de)DSB, with potentially divergent effects on repair pathway choice and genomic instability. Emerging evidence suggests that the biochemical environment of the broken mammalian replication fork may be specialized in such a way as to skew repair in favor of homologous recombination at the expense of non-homologous end joining.

Keywords: BRCA1; Break-induced replication; Homologous recombination; Mammalian DSB repair; One-ended break; Replication fork breakage.

Figure 1.. Canonical model of two-ended break formation at nickase-broken forks.

The collision of two converging replisomes with the nick site could generate two single-ended breaks (seDSBs) of opposite polarity, which are then repaired by two-ended homologous recombination.

Figure 2.. Mechanisms of one-ended break formation at nickase-broken forks.

Model derived from Vrtis et al. [32]. A. Leading strand replication fork collapse. CMG helicase association with the leading parental strand is lost following encounter with the nick, generating a single-ended break (seDSB). B. and C. Lagging strand replication fork collapse. Mode of translocation of CMG alters from ssDNA to dsDNA following CMG encounter with the free 5’ end of the ssDNA-dsDNA junction of the lagging strand nick. This leads to CMG ubiquitylation by CRL2^Lrr1, its extraction by p97 and degradation by the proteasome.

Figure 3.. CMG bypass of a Cas9^D10A-induced lagging strand nick.

Model derived from Pavani et al. [37].The CMG translocates along leading strand ssDNA displaced by binding of Cas9^D10A/sgRNA to its target site on the lagging strand. Crucially, the 5’ end of the ssDNA-dsDNA junction of the lagging strand nick is masked by the binding of Cas9^D10A/sgRNA, and CMG bypasses the nick, leaving a double-ended break (deDSB) on the lagging strand in the wake of the uninterrupted fork.