SSB Protein Limits RecOR Binding onto Single-stranded DNA

Michael D Hobbs,Akiko Sakai,Michael M Cox

doi:10.1074/jbc.m611007200

Michael D Hobbs, Akiko Sakai + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m611007200

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Abstract

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

Highlights

RecA carries out recombination reactions as a helical nucleoprotein filament [6]
Filament nucleation is followed by filament extension in the 5Ј to 3Ј direction along the DNA (9 –12)
Throughout, RecA-mediated hydrolysis of ATP is employed as an indirect measure of RecA protein binding to single-stranded DNA (ssDNA)

Summary

Introduction

RecA carries out recombination reactions as a helical nucleoprotein filament [6]. Formation of the active filament requires ATP and Mg2ϩ ion. Proteins that regulate RecA activity exert their control primarily by affecting the kinetics of filament formation and disassembly. The RecF protein, for example, antagonizes the activity of the RecX protein [26] These interactions provide a complex network for RecA filament control, the understanding of which is expanding as experiments progress [26, 36]. When SSB is prebound to ssDNA, it creates a significant kinetic barrier to RecA nucleation [27, 31] Overcoming this barrier is the function of the RMPs [18, 27, 31]. The E. coli RecF, RecO, and RecR proteins have all been implicated genetically in the nucleation of RecA filaments onto SSB-coated ssDNA. We have concentrated here on the function of the RecOR protein complex

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