Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection

Abstract

Double-strand breaks in chromosomal DNA are repaired efficiently in eukaryotic cells through pathways that involve direct religation of broken ends, or through pathways that utilize an unbroken, homologous DNA molecule as a template for replication. Pathways of repair that require homology initiate with the resection of the 5' strand at the break site, to uncover the 3' single-stranded DNA that becomes a critical intermediate in single-strand annealing and in homologous strand exchange. Resection of the 5' strand is regulated to occur most efficiently in S and G(2) phases of the cell cycle when sister chromatids are present as recombination templates. The mechanisms governing resection in eukaryotes have been elusive for many years, but recent work has identified the major players in short-range processing of DNA ends as well as the extensive resection of breaks that has been observed in vivo. This review focuses on the Mre11/Rad50/Xrs2(Nbs1) complex and the Sae2(CtIP) protein and their roles in initiating both short-range and long-range resection, the effects of topoisomerase-DNA conjugates on resection in vivo, and the relationship between these factors and NHEJ proteins in regulating 5' strand resection in eukaryotic cells.

Figure 1

Schematic diagram of proposed mechanisms by which the MRX complex and Sae2 facilitate the activity the downstream enzymes, shown as “Nuc”, to represent either Exo1 or Dna2(Sgs1/Rmi1/Top3) nucleases. See text for details. Recruitment (left panel): MRX and Sae2 promote nuclease binding to DNA by creating a higher-affinity binding site for the nuclease (here shown as a branched DNA structure). Nuclease bound to the MRX/Sae2-bound DNA makes an endonucleolytic cut (scissors) on the 5' strand. Further resection occurs by nucleases independently of MRX/Sae2. The event that triggers MRX and Sae2 dissociation is not known. End processing on unmodified ends (center panel, left side): MRX and Sae2 cleave the 5' strand of the DNA end through endonucleolytic activity (scissors); in the case of Exo1, this short 3' overhang facilitates nuclease binding and digestion. End processing on modified ends (center panel, right side): MRX and Sae2 cleave an adduct from the DNA (shown here as the 5' strand but in theory could be the 3' strand), allowing access of nucleases to the ends. Alternatively, Dna2 may also perform adduct cleavage [56]. Ku inhibition (right panel): the Ku heterodimer binds avidly to DNA ends; this occurs in competition with MRX binding. Some of the time MRX wins this competition and blocks Ku from binding; this allows loading of Exo1 and resection. Dna2 binding to DNA ends in vivo is relatively unaffected by Ku, while Exo1 binding is strongly affected [64], thus the nuclease in this case is likely to be Exo1. The exact configuration of the proteins on the DNA is not known, although Exo1 has been shown to crosslink to a DNA end on the 5' strand, while Sae2 crosslinks to the DNA on the 3' strand in strand-specific crosslinking experiments [25].