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Review
. 2016 Jan;26(1):52-64.
doi: 10.1016/j.tcb.2015.07.009. Epub 2015 Oct 1.

Repair Pathway Choices and Consequences at the Double-Strand Break

Raphael Ceccaldi  1 , Beatrice Rondinelli  1 , Alan D D'Andrea  2
Affiliations
Review

Repair Pathway Choices and Consequences at the Double-Strand Break

Raphael Ceccaldi et al. Trends Cell Biol. 2016 Jan.

Abstract

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

Keywords: DNA repair; Polθ; alternative end joining; homologous recombination; synthetic lethality.

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Figures

Figure 1
Figure 1. Four Approaches to Repair DNA Double-Strand Breaks (DSBs)
(A–D) The repair of DNA DSBs relies primarily on whether DNA end resection occurs. When resection is blocked, repair through C-NHEJ is favored. However, when DNA resection occurs, three pathways (HR, alt-EJ, and SSA) can compete for the repair of DSBs. Indeed, there are two layers of competition for the repair of DSBs. Initially at the stage of end resection, C-NHEJ competes with the resection-dependent pathways. Secondly, once resection has occurred, HR, alt-EJ, and SSA can compete for the repair. Notably, each of the four repair pathways lead to different genetic outcomes (LOH, deletions, insertions) and the fidelity of the repair mechanism is mentioned for each pathway. Abbreviations: nt, nucleotides; LOH, loss of heterozygosity; C-NHEJ, classical nonhomologous end joining; HR, homologous recombination; alt-EJ, alternative end joining; SSA, single-strand annealing.
Figure 2
Figure 2. Mechanisms Regulating DNA End Resection in Mammalian Cells and Their Influence on DNA Repair Pathway Choice
The cell cycle controls the competition between C-NHEJ and resection-dependent repair pathways. Extensive end resection is stimulated in the S/G2 phase of the cell cycle in a manner that depends on CDK activity, which mediates phosphorylation of multiple substrates, such as components of the MRN complex and CtIP. In the G1 phase of the cell cycle, 53BP1 and Rif1 proteins localize to DSBs, inhibit BRCA1 recruitment, block DNA end resection thus promoting C-NHEJ. In the S and G2 phases of the cell cycle, the ATM kinase, which phosphorylates members of the MRN complex, BRCA1, CtIP, or BLM favors the three resection-dependent DSB repair pathways (HR, alt-EJ, or SSA). However, recent evidence also suggests that alt-EJ could occur in G1 [105]. Abbreviations: ATM, Ataxia Telangiectasia Mutated; CDK, cyclin-dependent kinase; DSBs, double-strand breaks; MRN, MRE11, RAD50, and NBS1; C-NHEJ, classical nonhomologous end joining; HR, homologous recombination; alt-EJ, alternative end joining; SSA, single-strand annealing.
Figure 3
Figure 3. Regulation of RAD51-Mediated Recombination
During the HR process, once resection occurs, homology search and DNA-strand invasion generate D loops, a key intermediate for all subpathways of HR. This reaction catalyzed by RAD51 is negatively controlled at different levels. At the presynaptic level, Polυ prevents RAD51 nucleofilament formation. Postsynaptically, many proteins such as yeast Srs2 or human PARI, RECQL5, and BLM are able to dismantle formed RAD51 nucleofilaments. Additionally, HR can also be regulated at the level of strand exchange by RTEL1- or FANCM-mediated D-loop displacement. Concomitantly, players that negatively regulate HR promote DSB repair through alternative error-prone mechanisms such as alt-EJ or SSA. Abbreviations: CO, crossover; SDSA, synthesis-dependent strand annealing; TLS, translesion synthesis; SSA, single-strand annealing; alt-EJ, alternative end joining; DSB, double-strand break; HR, homologous recombination.
Figure 4
Figure 4. Polυ Regulates the Balance between Homologous Recombination (HR) and Alternative End Joining (alt-EJ)
(A) In the case of a HR defect, such as mutations in BRCA1/2 genes, Polυ is overexpressed. Polυ blocks the formation of RAD51–ssDNA nucleofilament and thus RAD51 toxicity in cells deficient in HR. (B) At the same time, Polυ is recruited to DNA damage sites by PARP1 where it performs untemplated error-prone synthesis required for alt-EJ. Polυ recruitment to damage sites might also depend on other alt-EJ factors (gray circles). Abbreviation: ssDNA, single-stranded DNA.
Figure 5
Figure 5. The Different Outcomes of Annealing-Dependent Error-Prone Double-Strand Break (DSB) Repair
After formation of a DSB, DNA resection results in two single-stranded overhangs and exposure of DNA homology. SSA uses annealing of large DNA sequences of homology, which leads to deletions of large fragments of DNA. By contrast, alt-EJ requires pairing of only small homologous DNA sequences (microhomologies), which can lead to deletions and/or insertions depending on how the repair is orchestrated: (i) When the annealing is stable the overhanging noncomplementary flaps are trimmed by the endonuclease complex and repair is completed by fill-in DNA synthesis and ligation, resulting in deletions of the DNA regions flanking the original break. (ii) Alternatively, translesion polymerases (such as Polυ) may extend the annealed sequences using untemplated error-prone DNA synthesis resulting in realignment at newly created micro-homologous sequences. Then the repair is completed by flap trimming, gap-filling DNA synthesis, and ligation, which results in a deletion plus insertion junction. (iii) Flap trimming followed by untemplated DNA error-prone synthesis can generate new regions of microhomology leading to insertions of DNA sequences. Abbreviations: alt-EJ, alternative end joining; DSB, double-strand break; SSA, single-strand annealing.
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