ATP Hydrolysis and DNA Binding by the Escherichia coli RecF Protein

Michael M Cox,Brian L Webb,Ross B Inman

doi:10.1074/jbc.274.22.15367

Michael M Cox, Brian L Webb + Show 1 more

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https://doi.org/10.1074/jbc.274.22.15367

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Abstract

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Highlights

Teins, acting together, limit the extension of RecA filaments beyond DNA gaps [15]
To determine whether less RecF protein is bound to the dsDNA in the presence of ATP as a result of ATP hydrolysis-induced dissociation, we examined the stability of these RecF1⁄7dsDNA complexes over time using a challenge experiment
Our primary conclusion is that ATP hydrolysis by RecF protein leads to its dissociation from dsDNA

Summary

Introduction

Teins, acting together, limit the extension of RecA filaments beyond DNA gaps [15]. All of the functions of RecF may come together in recombinational DNA repair, providing a useful paradigm within which to examine RecF protein activities [16, 17]. The RecF protein contains a consensus nucleotide binding fold (GXX GX GKT) in the N-terminal portion, which is highly conserved among the known recF gene sequences. The relevance of this nucleotide binding fold to the in vivo function of RecF was suggested by the observation that a single amino acid substitution within it (Lys to Arg at position 36) appears to be a null allele (recF4101) [18, 19]. Cells overexpressing the RecF4101 protein are viable [23] In light of these findings, we have studied the role of ATP hydrolysis in RecF protein function, focusing on DNA binding.

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