Abstract
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.
Highlights
The stalling of replication forks at sites of abnormal DNA structure, following collisions with transcription complexes or due to nucleotide pool depletion—collectively termed “replication stress”—is a significant contributor to genomic instability
We find that Rad51, a key mediator of homologous recombination (HR), accumulates in an intense and highly localized fashion at the stalled fork
We propose a model of HR initiation at the stalled fork in which a Rad51-mediated fork remodeling step prevents access of CNHEJ to the stalled fork
Summary
The stalling of replication forks at sites of abnormal DNA structure, following collisions with transcription complexes or due to nucleotide pool depletion—collectively termed “replication stress”—is a significant contributor to genomic instability. Inherited mutations in genes that regulate the replication stress response cause a number of human diseases, ranging from developmental disorders to highly penetrant cancer predisposition syndromes [1,2,3,4,5]. Replication fork stalling provokes a diverse set of cellular responses, including: stabilization of the stalled replisome; regulated replisome disassembly (“fork collapse”); protection of the fork from deleterious nucleolytic processing; remodeling of DNA structure at the stalled fork; and engagement of repair or “replication restart” [5, 7,8,9,10,11,12,13,14,15]. The mechanisms governing this coordination remain poorly understood in mammalian cells
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