The P. furiosus Mre11/Rad50 Complex Promotes 5′ Strand Resection at a DNA Double-Strand Break

Abstract

Summary The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro. The 3’ single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea, and may serve as a model for DSB processing in eukaryotes.

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

“…The yeast ortholog Mre11-Rad50-Xrs2 (MRX) cleaves the 5 0 strand approximately 15-25 nt from the DNA end in vitro in a reaction that requires ATP hydrolysis, Mre11's nuclease motif, and the Sae2/CtIP protein (Garcia et al, 2011;Cannavo & Cejka, 2014;Shibata et al, 2014). Endonucleolytic cleavage of DNA near the breaks has also been observed for both archaeal and bacterial homologs, and could play a role in deprotection of DNA ends in bacteriophage T4 recombination (Connelly et al, 2003;Hopkins & Paull, 2008;Almond et al, 2013). The precise mechanism of how ATP aids and regulates nucleolytic and structural functions in all of these reactions is not understood.…”

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

“…In vitro studies with purified proteins have also shown that MR and MRN(X) complexes recruit long range exo/endonucleases and also help to block the inhibitory effects of Ku (5,6,34,48,49). Here, we found that MRN strongly promotes DNA resection by Exo1 in the presence of both Ku and DNA-PKcs in a dose-dependent manner (Fig.…”

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

“…The canonical divalent cation for Mre11 is Mn 2ϩ , but recent work on Mre11 from Pfu and T4 indicate that Mg 2ϩ also supports nuclease activity (24,25). Interestingly, Mg 2ϩ appears to favor a potentially more physiologically relevant double-stranded DNA endonuclease activity that produces a 3Ј single-stranded DNA overhang that is an intermediate in the HR pathway (25).…”