ATP hydrolysis stimulates binding and release of single stranded DNA from alternating subunits of the dimeric E. coli Rep helicase: implications for ATP-driven helicase translocation.
DNA helicases are motor proteins that unwind duplex DNA during DNA replication, recombination and repair in reactions that are coupled to ATP binding and hydrolysis. In the process of unwinding duplex DNA processively, DNA helicases must also translocate along the DNA filament. To probe the mechanism of ATP-driven translocation by the dimeric E. coli Rep helicase along single stranded (ss) DNA, we examined the effects of ATP on the dissociation kinetics of ssDNA from the Rep dimer. Stopped-flow experiments show that the dissociation rate of a fluorescent ss oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ssDNA binding to the other subunit, and that the rate of this ssDNA exchange reaction is further stimulated approximately 60-fold upon ATP hydrolysis. This ssDNA exchange process occurs via an intermediate in which ssDNA is transiently bound to both subunits of the Rep dimer. These results suggest a rolling or subunit switching mechanism for processive ATP-driven translocation of the dimeric Rep helicase along ssDNA. Such a mechanism requires the extreme negative cooperativity for DNA binding to the second subunit of the Rep dimer, which insures that the doubly DNA-ligated Rep (P2S2) dimer is formed only transiently and relaxes back to the singly ligated Rep (P2S) dimer. The fact that other oligomeric DNA helicases share many functional features with the dimeric Rep helicase suggests that similar mechanisms for translocation and DNA unwinding may apply to other dimeric as well as hexameric DNA helicases.
Bjornson
K P
KP
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Wong
I
I
Lohman
T M
TM
D015075:2-Aminopurine; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001667:Binding, Competitive; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C506744:rep protein, E coli; D015075:2-Aminopurine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases