Repair of DNA double-strand breaks by mammalian alternative end-joining pathways

Abstract

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

Keywords: DNA damage; DNA endonuclease; DNA ligation; DNA polymerase; alternative end-joining pathway; chromosomes; deletions; double-strand break; genomic instability; microhomology; single-strand annealing; translocation.

Figure 1.

Role of DNA sequence homology in a-EJ pathways. Resection of the 5′ strand at DSBs is the first common step of all the EJ pathways (a-EJ). Three distinct pathways, single-strand annealing (SSA), microhomology-mediated end-joining (MMEJ), and end-joining (EJ) are distinguished based on the amount of DNA sequence complementarity used to align DNA ends. SSA involves complementary repeat sequences more than 25 nucleotides in length, whereas MMEJ involves shorter tracts of sequence homology, ranging from 2 to 20 nucleotides in length. There is also a third category of DSB repair events that either lack or have very little sequence homology at the repair site generated by a poorly defined EJ pathway.

Figure 2.

Repair of DSBs by the single-strand annealing pathway. 1) Introduction of a DSB break. 2) PARP-1 (PARP) mediates the rapid recruitment of MRN and CtIP to the DSB end. CtIP enhances the MRN endonuclease activity resulting in an internal single-strand break within the 5′ strand. The short single-strand fragment at the DSB end is then degraded by the MRN exonuclease activity. 3) The resultant single-strand region, which is rapidly bound by RPA, serves as the binding site for one of the processive 5′ to 3′ exonucleases, either Exo1 or DNA2. 4) The resultant long range resection by Exo1 or DNA2 exposes complementary single-strand regions, greater than 25 nucleotides in length. 5) Rad52 interacts with the RPA-coated single strands and anneals the complementary regions, aligning the DNA ends and exposing nonhomologous 3′ single-strand tails. 6) The single-strand tails are removed by ERCC1/XPF, a DNA structure-specific endonuclease that cleaves the 3′ strand at duplex/single-strand junctions. 7) After any gaps are filled, both strands are ligated to generate an intact duplex that is missing one of the repeats and the DNA region between the repeats. The DNA polymerases and DNA ligases involved in the last steps of SSA have not been identified.

Figure 3.

Repair of DSBs by the microhomology-mediated end-joining pathway. 1) Introduction of a DSB break. 2) PARP-1 (PARP) mediates the rapid recruitment of MRN and CtIP to the DSB end. CtIP enhances the MRN endonuclease activity resulting in an internal single-strand break within the 5′ strand. The short single-strand fragment at the DSB end is then degraded by the MRN exonuclease activity. 3) Short regions of sequence complementarity, ranging from 2 to 20 nucleotides, are exposed within the RPA-coated single-strand regions. 4) The DNA ends are transiently aligned via the short microhomologies. PARP-1, MRN, and Pol θ have each been implicated in the end alignment. It is likely that a similar, possibly MRN independent process of end alignment also occurs between ends that are being resected by the long-range exonucleases, Exo1 and DNA2 (see Fig. 2, step 3). 5) Nonhomologous 3′ tails are removed prior to error-prone gap-filling DNA synthesis by Pol θ. It is assumed that several functionally redundant nucleases will participate in end processing. 6) Both strands are ligated by the LigIIIα–XRCC1 complex (LigIII/XRCC1). 7) The DNA duplexes generated by MMEJ are characterized by deletions and the presence of sequence microhomologies at the repair site.