Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination.

Neville S Gilhooly,Carolina Carrasco,Benjamin Gollnick,Dale B Wigley,Mark S Dillingham,Fernando Moreno-Herrero,Martin Wilkinson

doi:10.1093/nar/gkv1543

Abstract

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.

Highlights

The repair of double-stranded DNA breaks by homologous recombination first requires resection of the DNA end to generate a long 3 -ssDNA overhang suitable for RecA/Rad51-dependent strand exchange
The B. subtilis AddAB helicase-nuclease complex catalyzes rapid and processive DNA end resection using a single Superfamily 1A (SF1A) helicase motor and two nuclease domains which are dedicated to the cleavage of each of the nascent unwound strands of DNA (2,6)
The resection of DNA breaks by wild type and mutant complexes was investigated with DNA substrates that either did, or did not, contain Chi sequences in defined positions

Summary

Introduction

The repair of double-stranded DNA breaks by homologous recombination first requires resection of the DNA end to generate a long 3 -ssDNA overhang suitable for RecA/Rad51-dependent strand exchange. The B. subtilis AddAB helicase-nuclease complex catalyzes rapid and processive DNA end resection using a single Superfamily 1A (SF1A) helicase motor and two nuclease domains which are dedicated to the cleavage of each of the nascent unwound strands of DNA (2,6). Continued unwinding and degradation of the 5 -terminated strand by the AddB nuclease domain produces a long 3 -ssDNA overhang; the substrate required for RecA-mediated strand exchange (9). This resection reaction is analogous to that performed by the well-studied RecBCD enzyme, there are significant differences between the two systems (see (2,10) for reviews)

Methods

Results

Conclusion