Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Susan K Amundsen,Gerald R Smith,Andrew F Taylor

doi:10.1038/s41598-020-73078-0

Abstract

Repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5′ GCTGGTGG 3′) recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, > 25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi hotspot-deficient mutant in the RecB helicase domain located > 45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path about 185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.

Highlights

Regulation of complex enzymes and reaction pathways is critical for all living organisms, because having too much or too little enzymatic activity, or having spurious activity, can be highly deleterious or lethal
Crystal and cryoEM structures of RecBCD bound to DNA show that, when the 3′-ended strand emerges from the RecB helicase, it enters a tunnel in RecC
Later research showed that one, recC343, changes proline 666 to leucine (P666L; 5′ CCA 3′ changed to 5′ CTA 3′), which is on the surface of the RecC tunnel in which Chi is recognized[14,29]

Summary

Introduction

Regulation of complex enzymes and reaction pathways is critical for all living organisms, because having too much or too little enzymatic activity, or having spurious activity, can be highly deleterious or lethal This principle holds true for the repair of broken DNA, which requires in many cases large, complex enzymes. In many bacteria, including Escherichia coli studied here, the major pathway of homologous recombination and DNA break repair requires RecBCD enzyme, a three subunit, 330 kDa enzyme with DNA unwinding (helicase) and DNA cutting (nuclease) activities (reviewed in[1]). Upon encountering Chi, the RecB nuclease cuts the emerging 3′-ended strand a few nucleotides 3′ of the Chi octamer, and the enzyme begins loading RecA DNA strand-exchange protein onto the newly generated 3′-ended strand[19,21,22]. The three subunits dissociate after cutting at Chi, likely upon leaving the DNA (at the end of linear DNA substrates with purified enzyme)[20]

Methods

Results

Conclusion