DNA end resection: many nucleases make light work

Abstract

Double-strand breaks (DSBs) are deleterious DNA lesions and if left unrepaired result in severe genomic instability. Cells use two main pathways to repair DSBs: homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the phase of the cell cycle and the nature of the DSB ends. A key step where pathway choice is exerted is in the 'licensing' of 5'-3' resection of the ends to produce recombinogenic 3' single-stranded tails. These tails are substrate for binding by Rad51 to initiate pairing and strand invasion with homologous duplex DNA. Moreover, the single-stranded DNA generated after end processing is important to activate the DNA damage response. The mechanism of end processing is the focus of this review and we will describe recent findings that shed light on this important initiating step for HR. The conserved MRX/MRN complex appears to be a major regulator of DNA end processing. Sae2/CtIP functions with the MRX complex, either to activate the Mre11 nuclease or via the intrinsic endonuclease, in an initial step to trim the DSB ends. In a second step, redundant systems remove long tracts of DNA to reveal extensive 3' single-stranded tails. One system is dependent on the helicase Sgs1 and the nuclease Dna2, and the other on the 5'-3' exonuclease Exo1.

Figure 1. Models for DSB repair by homologous recombination

The first and common step in all mechanisms is the 5′ to 3′ degradation of the DSB ends, to generate to invasive 3′ ssDNA tails (arrowhead). In the DSBR pathway, after priming DNA synthesis (dashed lines), second end capture allows formation of a double Holliday junction, whose resolution can lead to either crossover or non-crossover products. In the SDSA pathway, the 3′ end is displaced, it pairs with the other 3′ single stranded tail and DNA synthesis completes repair. The SSA pathway is restricted to DSBs that occur between direct repeats. Extensive resection of the ends reveals the complementary sequences, which can then anneal resulting in the deletion of one of the repeats and the intervening sequences.

Figure 2. Bridging of DSB ends by the Mre11-Rad50-Xrs2 complex

Each pentameric complex is comprised of a Rad50 dimer, an Mre11 dimer and an Xrs2 monomer. The apex of the Rad50 coiled-coil, where the Cys-X-X-Cys motif resides, provides the dimerization interphase via cysteine-mediated zinc ion coordination. Mre11 binds to the base of the coiled coil near the Rad50 DNA binding region. The Xrs2 monomer associates with the complex via interaction with the Mre11 dimer. Intermolecular dimerization between two individual Mre11₂Rad50₂Xrs2₁ complexes tethers the two ends of a DSB.

Figure 3. Two step mechanism of DSB resection in meiosis and mitosis

A. After meiotic DSBs are formed, Spo11-bound ends are poor substrates for Exo1 or Sgs1-Dna2, but when processed by MRX-Sae2 to remove Spo11 and form short 3′ overhangs they can be used by either Exo1 or Sgs1-Dna2. Whether Sgs1-Dna2 functions in processing meiotic DSBs remains to be determined. B. In G1, when Sae2 is not phosphorylated by CDK, the ends are preferentially bound by Ku70-Ku80 channeling repair to the NHEJ pathway. In G2, activation of Sae2 by CDK, leads to trimming of DSB ends, dissociation of the MRX complex from the DSB ends and generation of short RPA bound ssDNA overhangs. This trimming step could potentially inhibit Ku70-Ku80 binding and instead promote extensive resection by Sgs1-Dna2 or Exo1.