DNA nicks in both leading and lagging strand templates can trigger break-induced replication

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair Mechanisms

Background:

• Replication forks encountering unrepaired DNA single-strand breaks (SSBs) can lead to double-strand breaks (DSBs), including single-ended (seDSBs) and double-ended (deDSBs).
• Break-induced replication (BIR) is a mutagenic pathway implicated in cancer development, but its frequency and triggers remain incompletely understood.
• The roles of leading vs. lagging template strand SSBs in DSB formation and repair pathway choice require further investigation.

Purpose of the Study:

• To investigate the frequency and determinants of break-induced replication (BIR) following encounters between replication forks and DNA single-strand breaks (SSBs).
• To determine if leading and lagging template strand SSBs differentially trigger BIR.
• To explore the impact of replication fork convergence and non-homologous end joining (NHEJ) proteins on BIR.

Main Methods:

• Site- and strand-specific SSBs were generated in fission yeast using nicking enzymes, including CRISPR-Cas9 nickase (Cas9n).
• The repair outcomes of these SSBs, specifically conversion to deDSBs or triggering of BIR, were analyzed.
• Experiments involved manipulating fork convergence timing and deleting the non-homologous end joining protein Ku70.

Main Results:

• Leading and lagging template strand SSBs were typically converted into deDSBs repaired by homologous recombination.
• Both types of SSBs were capable of triggering BIR, a mutagenic DNA repair pathway.
• The frequency of BIR events increased significantly when replication fork convergence was delayed or when Ku70 was deleted.

Conclusions:

• DNA single-strand breaks (SSBs) encountered by replication forks can be repaired via homologous recombination or lead to mutagenic break-induced replication (BIR).
• Both leading and lagging strand SSBs can initiate BIR, with increased frequency under conditions of replication stress (delayed fork convergence) or impaired non-homologous end joining (Ku70 deletion).
• These findings provide critical insights into the mechanisms driving genome instability and cancer development.