E. coli Rep oligomers are required to initiate DNA unwinding in vitro.
E. coli Rep protein is a 3' to 5' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.
Cheng
W
W
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Hsieh
J
J
Brendza
K M
KM
Lohman
T M
TM
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D005456:Fluorescent Dyes; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases