RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.
The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.
Kim
Jong-Il
JI
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Sharma
Ajay K
AK
Abbott
Stephen N
SN
Wood
Elizabeth A
EA
Dwyer
David W
DW
Jambura
Aaron
A
Minton
Kenneth W
KW
Inman
Ross B
RB
Daly
Michael J
MJ
Cox
Michael M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006095:Gram-Positive Cocci / Q000737:chemistry* / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011938:Rec A Recombinases / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; C026600:2'-deoxyadenosine triphosphate