Resolution of collapsed forks is separate from completion of DNA synthesis

Abstract

Replication fork collapse at single-strand DNA breaks (SSBs) poses a serious threat to genome stability. Using Xenopus egg extracts, we show that a replication fork encountering an SSB on either the leading- or lagging-strand template produces a single-ended double-strand break (seDSB). These broken ends are efficiently resolved by homologous recombination to yield D-loops and erroneous end-to-end fusions. Surprisingly, DNA synthesis downstream of an seDSB is highly inefficient. In contrast, when two forks converge at an SSB, they generate a double-ended DSB (deDSB) that efficiently completes DNA synthesis through double-strand break repair that is not dependent on homologous recombination. Leading, but not lagging, seDSBs can undergo extensive nucleolytic degradation that disassembles the divergent fork. These secondary collapse events efficiently resolve seDSBs but without completion of DNA synthesis. Moreover, PARP inhibition can enhance fork collapse at unmodified SSBs but not at abasic site SSBs, contrary to expectations. Our findings distinguish end resolution from replication completion and demonstrate flexibility in how PARP inhibition affects fork collapse.