Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II.
T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.
Wessel
R
R
Max-Planck-Institut für Medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, Federal Republic of Germany.
Müller
H
H
Hoffmann-Berling
H
H
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000494:pharmacology* / Q000648:ultrastructure; D004247:DNA / Q000648:ultrastructure; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000737:chemistry* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology*; D005092:Exonucleases / Q000494:pharmacology; D008854:Microscopy, Electron; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D013604:T-Phages / Q000201:enzymology*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004247:DNA; D005092:Exonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases