Mechanism and regulation of DNA end resection in eukaryotes

Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5'-terminated strands in a process termed end resection. End resection generates 3'-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5'-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3'-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome.

Keywords: DNA repair; Dna2; Exo1; Mre11; Sae2/CtIP; double-strand break; end joining; recombination.

Figure 1. Models for the repair of DSBs by NHEJ, MMEJ, SSA or HR

The direct ligation of ends by NHEJ is favored in G1 phase cells when end resection activity is low. Activation of end resection in S-G2 phase cells creates 3′-ssDNA overhangs that are initially bound by RPA, and then by Rad51 to catalyze homologous pairing and strand invasion (HR). Alternatively, annealing of microhomologies internal to the ends results in repair by MMEJ, or by SSA if longer direct repeats flank the DSB. Rad1-Rad10 nuclease (human XPF-ERCC1) cleaves the 3′ flaps formed by annealing of repeats internal to the ends. Thick orange lines represent direct repeats flanking the DSB.

Figure 2. Activity of MRX at protein-bound and hairpin-capped ends

A. Phosphorylated Sae2 activates the Mre11 subunit of the MRX complex to cleave the 5′-terminated strand internal to the protein-bound end (Spo11 in meiotic cells). The nick acts as an entry site for the 3′-5′ Mre11 exonuclease and 5′-3′ Exo1 exonuclease for bidirectional resection. B. MRX can cleave the unpaired region of a DNA hairpin without Sae2, or potentially Sae2 could activate MRX cleavage at a distance from the end as proposed for protein-bound ends.

Figure 3. Resection initiation in the absence of Mre11 nuclease, Sae2 or Ku

Resection initiation at DSBs generated by endonucleases can occur by MRX-Sae2 cleavage, as envisioned for protein-bound ends (left panel). In the absence of the Mre11 nuclease or Sae2, Dna2-STR is the main nuclease for resection initiation, unless Ku is absent (middle panel). Exo1 can initiate resection from ends when Ku is eliminated (right panel).

Figure 4. DNA damage checkpoint regulation of end resection

MRX activates Tel1, which phosphorylates Sae2 and H2A. RPA binds to the ssDNA tail formed by MRX-Sae2 clipping and recruits Dna2-STR and Mec1-Ddc2. Mec1-Ddc2 is mainly responsible for phosphorylation of Rad9, Rad53 and H2A. The PCNA-like 9-1-1 complex binds at the ssDNA-dsDNA transition and activates Exo1 and Dna2. Rad9 binds to modified histones preventing extensive resection by Exo1 and Dna2. Rad9 also activates Rad53, which phosphorylates Exo1 to inhibit its activity.