A Possible Role of the C-terminal Domain of the RecA Protein: A GATEWAY MODEL FOR DOUBLE-STRANDED DNA BINDING

Hitoshi Kurumizaka,Hideki Aihara,Shukuko Ikawa,Takamitsu Kashima,L Rochelle Bazemore,Katsumi Kawasaki,Akinori Sarai,Charles M Radding,Takehiko Shibata

doi:10.1074/jbc.271.52.33515

Abstract

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270-328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in double-stranded DNA binding and that they constitute a part of a gateway for homologous recognition.

Highlights

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270 –328
We introduced amino acid substitutions in the domain consisting of amino acid residues 270 –328, a candidate for the gateway for secondary binding of double-stranded DNA to presynaptic filaments of RecA protein and single-stranded DNA
We looked at basic amino acid residues (Lys or Arg) that could directly interact with negatively charged sugar phosphate backbones of DNA

Summary

Introduction

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270 –328 (from the N terminus). An x-ray crystallographic study and features of mutant RecA proteins suggest that the DNA binding sites are the regions from the 157th to 164th amino acid residues (called loop 1) and from the 195th to 209th amino acid residues (called loop 2), each of which forms a disordered loop (13 and references therein for mutations). This view was supported by photochemical cross-linking experiments [14, 15]. These observations further support the hypothesis that loop 2 is a DNA binding site critical for homologous pairing

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