A chimeric RecA protein exhibits altered double-stranded DNA binding.
RecAc38 protein, a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, is proficient in the renaturation from complementary single strands. However, RecAc38 protein showed a significant deficiency in promoting homologous pairing of single-stranded DNA and double-stranded DNA. RecAc38 protein was able to remove the secondary structure of single-stranded DNA, the first step of homologous pairing, at a slightly reduced rate. RecAc38 protein-single-stranded DNA-complex (presynaptic complex) was found to be deficient in the sequence-independent binding to double-stranded DNA which is a step in the search for the homology. On the other hand, once unwinding was initiated, RecAc38 protein was able to propagate the unwinding of the double helix at the same extent as wild-type RecA protein. These defects and the proficiency of RecAc38 protein are explained by a model showing that RecA protein has three distinct DNA strand-binding sites (a site for the primary binding to single-stranded DNA, a site for the binding to a strand of second single- or double-stranded DNA, and a site required for the binding to the other strand of the double-stranded DNA) and that RecAc38 protein has a defect in the third site.
Kurumizaka
H
H
Laboratory of Cellular & Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Ikawa
S
S
Ikeya
T
T
Ogawa
T
T
Shibata
T
T
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases