A Possible Role of the C-terminal Domain of the RecA Protein

doi:10.1074/jbc.271.52.33515

A Possible Role of the C-terminal Domain of the RecA Protein

A GATEWAY MODEL FOR DOUBLE-STRANDED DNA BINDING*

From the ^‡ Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan,
^§ Graduate School of Science and Engineering, Saitama University, Urawa-shi, Saitama 338, Japan,
^∥ Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113, Japan,
** Department of Genetics and Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510, and
^‡‡ Gene Bank, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (Rikagaku Kenkyū-jo), 3-1-1 Koyadai, Tsukuba 305, Japan

^¶¶To whom correspondence should be addressed. Fax: 81-48-462-4671; E-mail: tshibata{at}postman.riken.go.jp.

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.

Footnotes

↵^¶ Supported by a fellowship from the Japan Society for the Promotion of Science.
↵^§§ Supported by Grant RO1-GM33504 from the National Institutes of Health.
↵* This study was partly supported by a grant for the Biodesign Research Program from Rikagaku Kenky-jo and a grant from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵³ The abbreviations used are:

PIPES

1,4-piperazinediethanesulfonic acid

SSB

single-stranded DNA-binding protein

ATPγS

adenosine 5′-O-(thiotriphosphate).
↵¹ A. Sarai and H. Kurumizaka, unpublished model building study.
↵² T. Kashima and K. Kawasaki, unpublished observation.
↵⁴ S. Ikawa, O. Makino, and T. Shibata, unpublished observation.
↵⁵ N. Arai and K. Kawasaki, unpublished observation.
- Received June 18, 1996.
- Revision received October 7, 1996.