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DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required.
- Source :
-
Biochemistry [Biochemistry] 2000 May 30; Vol. 39 (21), pp. 6401-9. - Publication Year :
- 2000
-
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.
- Subjects :
- Hydrolysis
Kinetics
Magnesium metabolism
Models, Chemical
Oligodeoxyribonucleotides chemistry
Spectrometry, Fluorescence
Thymidine Monophosphate metabolism
Thymine Nucleotides metabolism
Bacteriophage T7 enzymology
DNA Primase chemistry
DNA Primase metabolism
DNA, Single-Stranded chemistry
DNA, Single-Stranded metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 39
- Issue :
- 21
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10828954
- Full Text :
- https://doi.org/10.1021/bi992857i