title: Electron microscopy of DNA.helicase-I complexes in the act of strand separation.
abstract: Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB-complexed DNA were observed as forks. The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5'-3' direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand.
authors.affiliation: Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, Federal Republic of Germany.
authors.forename: R
authors.initials: R
authors.lastname: Wessel
authors.forename: H
authors.initials: H
authors.lastname: Müller
authors.forename: H
authors.initials: H
authors.lastname: Hoffmann-Berling
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D003090:Coliphages / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C027093:TraI protein, E coli; D004265:DNA Helicases