The repair of chromosomal double strand breaks (DSBs) is crucial in the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability1–3. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobasepairs of DNA from a donor molecule all the way through its telomere4,5. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis4–9. BIR employs the majority of known replication proteins to copy large portions of DNA, similar to S-phase replication10,11. It has thus been suggested that BIR proceeds by semiconservative replication; however, the model of a bona-fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1000-fold increase in mutation rate compared to normal replication9. Here we demonstrate that the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide the evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the dramatic increase in BIR-associated mutations. We propose that the BIR-mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes including humans.

SUMMARY RAG initiates antibody V(D)J recombination in developing lymphocytes by generating “on-target” DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically-inserted RSS pairs to identify huge numbers of RAG “off-target” breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage-site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation-dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJ intermediate formation by isolating Ds, Js, and RCs from upstream Vs in a chromatin loop anchored by CTCF-binding elements (CBEs). How Vs access the DJRC for V to DJ rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged V-RSSs increase recombination potential of their associated V far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. V-associated CBEs stabilize interactions of D-proximal Vs first encountered by the DJRC during linear RAG scanning and thereby promote dominant rearrangement of these Vs by an unanticipated chromatin accessibility-enhancing CBE function.