Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier

Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.Author summary: Genomic instability is a significant contributor to human disease, ranging from hereditary developmental disorders to cancer predisposition. Two major triggers to genomic instability are chromosomal double strand breaks (DSBs) and the stalling of replication forks during the DNA synthesis (S phase) of the cell cycle. The “rules” that govern mammalian DSB repair are increasingly well understood, and it is recognized that the two major DSB repair pathways—classical non-homologous end joining (C-NHEJ) and homologous recombination (HR)—compete to repair a mammalian DSB. In contrast, we do not yet have equivalent insight into the regulation of repair at sites of mammalian replication fork stalling. Here, we explore the relationship between C-NHEJ and HR at a defined chromosomal replication fork barrier in mammalian cells. We show that, in contrast to DSB repair, repair at stalled forks does not entail competition between C-NHEJ and HR. We find that Rad51, a key mediator of HR, accumulates in an intense and highly localized fashion at the stalled fork. Based upon these findings, we propose a model of HR initiation at the stalled fork in which a Rad51-mediated fork remodeling step prevents access of C-NHEJ to the stalled fork.