Error-Prone Repair of DNA Double-Strand Breaks

Abstract

Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double-strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non-homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error-free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error-prone double-strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders.

Figure 1. Mutagenic potential of different double-strand break repair mechanisms

Double-strand breaks can be repaired via either non-homologous end joining (NHEJ) or homologous recombination (HR) mechanisms. While classical NHEJ (C-NHEJ) can result in perfect repair, small insertions/deletions are also possible. Microhomology-mediated end joining (MMEJ) is a deletional repair mechanism, while other forms of alternative end joining (A-NHEJ) always result in changes to the DNA sequence. Homologous recombination that proceeds via double Holliday junction intermediates (DSBR) or synthesis-dependent strand annealing (SDSA) are potentially mutagenic, especially if template switching occurs, while break-induced replication (BIR) is highly mutagenic and can lead to complex chromosomal aberrations.

Figure 2. Mechanisms and outcomes of Non-Homologous End Joining (NHEJ)

A. Creation of a DNA double-stranded break. B. Repair via microhomology-mediated end joining (MMEJ). The break is resected (or ends are unwound) and exposed microhomologous sequences anneal (vertical lines). Repair is completed by flap removal, fill-in synthesis, and DNA ligation. C. Repair by classical non-homologous end joining (C-NHEJ). Binding of the Ku heterodimer to DNA ends protects the DNA from extensive resection or unwinding. If necessary, processing enzymes such as nucleases and polymerases are recruited. Completion of repair depends on DNA Ligase 4. Green hourglass represents the protein complex (Ku plus other proteins) that synapse and process the DNA ends. D. Repair by polymerase theta-mediated end joining (TMEJ). Short regions of homology are extended by Polθ. If no further processing occurs, the result is a deletion (not shown). Following synthesis, multiple rounds of unwinding, reannealing, and Polθ-dependent synthesis can lead to the addition of templated insertions resulting in simple deletions. Repair outcomes of additional small templated insertions (represented as red, dashed lines).

Figure 3. Opportunities for mutagenesis during homologous recombination repair of a DNA double-strand break

A. Creation of a DNA double-strand break. B. Resection of broken ends creates 3’ single-stranded DNA that can be easily damaged. C. One-ended strand invasion into a homologous template creates a displacement (D)-loop. Synthesis during D-loop extension is potentially mutagenic. Initial strand invasion can also occur at homeologous or microhomologous sequences, resulting in insertion/deletion repair products. D. During synthesis-dependent strand annealing (SDSA), D-loop dissociation and annealing of the nascent strand with single-stranded DNA from the broken chromosome is followed by potentially mutagenic single-stranded gap filling and ligation. E. Alternatively, two-ended invasion and synthesis leads to double Holliday junction (dHJ) formation. Resolution of the dJHs can create crossover (shown) or non-crossover (not shown) products. Asterisks indicate new potential sites of mutagenesis at each step.

Figure 4. Opportunities for mutagenesis during break-induced replication (BIR)

A. Creation of a one-ended double-strand break. B. Resection of broken ends creates 3’ single-stranded DNA (ssDNA) that can be easily damaged. C. One-ended strand invasion into a homologous template creates a displacement (D)-loop. Synthesis during D-loop extension in BIR is highly mutagenic. D. Migration of the D-loop results in extensive accumulation of ssDNA. Unwinding of the D-loop prior to completion of synthesis and re-invasion into homeologous or microhomologous sequences can result in insertion/deletion mutations. E. Processive synthesis up to hundreds of kilobases to the end of a chromosome and accumulation of more ssDNA, which is easily damaged. F. Completion of repair via lagging strand synthesis. G. DNA damage or genetic impairment of BIR can stall repair synthesis. H. Subsequent resolution of the blocked replication fork results in a half crossover (HCO) event. I. Re-invasion of the broken template can result in subsequent rounds of BIR. Asterisks indicate new potential sites of mutagenesis at each successive step.