RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps

E A Ronayne,M M Cox

doi:10.1093/nar/gkt1342

Abstract

The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.

Highlights

Recombination enhancement function (Ref) is a 21-kDa RecA-dependent endonuclease encoded by the temperate bacteriophage P1 [1]
Ref endonuclease activity can be restricted to displacement loops (D-loops) created by the RecA protein [1]
When a D-loop is created in supercoiled double-stranded DNA (dsDNA), Ref cleaves one strand of the D-loop, resulting in nicked circular dsDNA (cdsDNA), or both strands, creating a 7.25-kb linear duplex (Figure 1A)

Summary

Introduction

Recombination enhancement function (Ref) is a 21-kDa RecA-dependent endonuclease encoded by the temperate bacteriophage P1 [1]. Transcription of ref is under the control of the phage P1 c1 master repressor, suggesting that ref transcription plays a role in the lytic cycle [5]. This repressor functions in an autoregulatory control loop, binding 22 sites on the P1 genome. The linear double-stranded P1 genome must be cyclized to avoid degradation by host exonucleases. This process occurs either by RecA-dependent homologous recombination of terminally redundant ends, or by phage-encoded Cre-lox site-specific recombination [6]. Ref may play a role in RecA-dependent cyclization, and the maintenance of P1 as a low copy number plasmid [7]

Methods

Results

Conclusion