The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing.
In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.
Kurumizaka
H
H
Cellular & Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Saitama, Wako-shi, 351-0198, Japan.
Ikawa
S
S
Sarai
A
A
Shibata
T
T
D020029:Base Pairing; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays / Q000009:adverse effects
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA; D011938:Rec A Recombinases