A possible role of the C-terminal domain of the RecA protein. A gateway model for double-stranded DNA binding.
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.
Kurumizaka
H
H
Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan. tshibata.postman.riken.go.jp
Aihara
H
H
Ikawa
S
S
Kashima
T
T
Bazemore
L R
LR
Kawasaki
K
K
Sarai
A
A
Radding
C M
CM
Shibata
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry*
D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D011938:Rec A Recombinases; D008239:Lysine