Break-induced replication (BIR) is a pathway that repairs one-ended double-strand breaks (DSBs). For decades, yeast model systems offered the only opportunities to study eukaryotic BIR. These studies described an unusual mode of BIR synthesis that is carried out by a migrating bubble and shows conservative inheritance of newly synthesized DNA, leading to genomic instabilities like those associated with cancer in humans. Yet, evidence of BIR functioning in mammals or during repair of other DNA breaks has been missing. Recent studies have uncovered multiple examples of BIR working in replication restart and repair of eroded telomeres in yeast and mammals, as well as some unexpected findings, including the RAD51 independence of BIR. Strong interest remains in determining the variations in molecular mechanisms that drive and regulate BIR in different genetic backgrounds, across organisms, and particularly in the context of human disease.

Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Break-induced replication (BIR) repairs one-ended double strand breaks (DSBs) similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human disease 1 , 2 . Previous studies have defined the enzymes required for BIR 1 – 5 ; however, understanding of initial and extended BIR synthesis as well as how the migrating D-loop proceeds through known replication roadblocks has been precluded by technical limitations. Here, using a newly developed assay, we demonstrate that BIR synthesis initiates soon after strand invasion and proceeds slower than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but fails to proceed beyond 30 kb, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32 but does not proceed efficiently. We demonstrate that interstitial telomeric DNA disrupts and terminates BIR progression. Also, BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and on how BIR contributes to genome instability.

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.

Extrachromosomal circular DNA (eccDNA) originates from linear chromosomal DNA in various human tissues under physiological and disease conditions. The genomic origins of eccDNA have largely been investigated using in vitro–amplified DNA. However, in vitro amplification obscures quantitative information by skewing the total population stoichiometry. In addition, the analyses have focused on eccDNA stemming from single-copy genomic regions, leaving eccDNA from multicopy regions unexamined. To address these issues, we isolated eccDNA without in vitro amplification (naïve small circular DNA, nscDNA) and assessed the populations quantitatively by integrated genomic, molecular, and cytogenetic approaches. nscDNA of up to tens of kilobases were successfully enriched by our approach and were predominantly derived from multicopy genomic regions including segmental duplications (SDs). SDs, which account for 5% of the human genome and are hotspots for copy number variations, were significantly overrepresented in sperm nscDNA, with three times more sequencing reads derived from SDs than from the entire single-copy regions. SDs were also overrepresented in mouse sperm nscDNA, which we estimated to comprise 0.2% of nuclear DNA. Considering that eccDNA can be integrated into chromosomes, germline-derived nscDNA may be a mediator of genome diversity.

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Microhomology-mediated break-induced replication (MMBIR) is a DNA repair pathway initiated by polymerase template switching at microhomology, which can produce templated insertions that initiate chromosomal rearrangements leading to neurological and metabolic diseases, and promote complex genomic rearrangements (CGRs) found in cancer. Yet, how often templated insertions accumulate from processes like MMBIR in genomes is poorly understood due to difficulty in directly identifying these events by whole genome sequencing (WGS). Here, by using our newly developed MMBSearch software, we directly detect such templated insertions (MMB-TIs) in human genomes and report substantial differences in frequency and complexity of MMB-TI events between normal and cancer cells. Through analysis of 71 cancer genomes from The Cancer Genome Atlas (TCGA), we observed that MMB-TIs readily accumulate de novo across several cancer types, with particularly high accumulation in some breast and lung cancers. By contrast, MMB-TIs appear only as germline variants in normal human fibroblast cells, and do not accumulate as de novo somatic mutations. Finally, we performed WGS on a lung adenocarcinoma patient case and confirmed MMB-TI-initiated chromosome fusions that disrupted potential tumor suppressors and induced chromothripsis-like CGRs. Based on our findings we propose that MMB-TIs represent a trigger for widespread genomic instability and tumor evolution.

Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.