DNA resection in eukaryotes: deciding how to fix the break

Abstract

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation.

Figure 1

The repair of DNA double-strand breaks (DSBs). DSBs can be repaired using several different mechanisms. Both ends can be simply rejoined with little or no further processing (nonhomologous end-joining; NHEJ) or can be repaired using homologous sequences (red DNA; homologous recombination) after 5′–3′ degradation has occurred (resection). The 3′-OH group exposed after resection can be used to prime DNA synthesis using a homologous region as a template after DNA strand invasion. The newly synthesized DNA (light blue) can then be joined with the 5′ end of the resected strand forming a double Holliday junction (double-strand break repair; DSBR), or can be displaced and reannealed (synthesis-dependent strand annealing; SDSA); or DNA synthesis can continue to the end of the chromosome (break-induced replication; BIR). If two homologous regions flank the DSB, they can anneal after being exposed by DNA resection (single-strand annealing; SSA), which causes the deletion of the intervening region. An additional mechanism that shares components with both SSA and NHEJ, and uses short homology stretches (usually 2–3 bp) flanking the DSB, can also be used (microhomology-mediated end-joining; MMEJ). The chromosomal consequences of aberrant use of each repair mechanism are shown in the red boxes.

Figure 2

Mechanism of resection in budding yeast. DSBs are detected by the Mre11 complex (MRX) and Sae2. Upon activation of the endonucleolytic activity of MRX and Sae2, initial processing results in the generation of short stretches of single-stranded DNA. This partially resected DNA will then be the substrate for further nucleolytic degradation either by Exo1 or by Dna2 and Sgs1. The initial processing by Mre11 and Sae2 can be bypassed in mitotic interphase (dashed black arrow), probably by the action of Exo1 or of Sgs1 and Dna2. In the absence of Exo1 and Sgs1, several rounds of the endonucleolytic activity of Mre11-Sae2 will be sufficient for short processing close to the ends (dashed red arrow).

Figure 3

Regulation of resection in budding yeast. Schematic representation of how DNA-end resection is regulated. Positive actions are shown as red arrows and negative regulations as light blue arrows; solid arrows represent interactions by known mechanisms and dashed arrows interactions by unknown mechanisms. Question marks indicate points at which additional layers of regulation may be acting. DNA end resection is activated in S/G2 cells by the activity of CDKs, directly by phosphorylation of Sae2 and by an unknown mechanism regulating Rad9. Although Rad9 does not bind naked DNA but rather chromatin, histones are not shown for simplification. The presence of KU and Rad9 are negative regulators of resection. Multiple or ‘ragged’ ends also stimulates DNA processing even in G1 cells.