title: DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required.
abstract: Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Picha
authors.forename: P
authors.initials: P
authors.lastname: Ahnert
authors.forename: S S
authors.initials: SS
authors.lastname: Patel
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013938:Thymidine Monophosphate / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D013942:Thymine Nucleotides; D013938:Thymidine Monophosphate; D019915:DNA Primase; D008274:Magnesium; C024157:thymidine 5'-triphosphate