Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA.
RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.
Slocum
Stephen L
SL
Department of Biochemistry, University at Buffalo, Buffalo, NY 14214, USA.
Buss
Jackson A
JA
Kimura
Yuji
Y
Bianco
Piero R
PR
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006493:Heparin; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011071:Poly T / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; C071446:RecG protein, E coli; D011071:Poly T; D000255:Adenosine Triphosphate; D006493:Heparin; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium