Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed
- PMID: 10625495
- DOI: 10.1021/bi992105o
Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed
Abstract
Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.
Similar articles
-
The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.J Mol Biol. 2009 Oct 2;392(4):1020-32. doi: 10.1016/j.jmb.2009.07.071. Epub 2009 Jul 30. J Mol Biol. 2009. PMID: 19647000
-
Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.Biochemistry. 2002 Jan 15;41(2):643-51. doi: 10.1021/bi011137k. Biochemistry. 2002. PMID: 11781105
-
Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.EMBO J. 2000 Jul 17;19(14):3799-810. doi: 10.1093/emboj/19.14.3799. EMBO J. 2000. PMID: 10899133 Free PMC article.
-
Fluorescence tools to measure helicase activity in real time.Methods. 2010 Jul;51(3):259-68. doi: 10.1016/j.ymeth.2010.02.012. Epub 2010 Feb 16. Methods. 2010. PMID: 20167273 Review.
-
DNA helicases: 'inching forward'.Curr Opin Struct Biol. 2000 Feb;10(1):124-8. doi: 10.1016/s0959-440x(99)00059-7. Curr Opin Struct Biol. 2000. PMID: 10679457 Review.
Cited by
-
The Role of SF1 and SF2 Helicases in Biotechnological Applications.Appl Biochem Biotechnol. 2024 Aug 2. doi: 10.1007/s12010-024-05027-w. Online ahead of print. Appl Biochem Biotechnol. 2024. PMID: 39093351 Review.
-
Multiscale simulations reveal the role of PcrA helicase in protecting against spontaneous point mutations in DNA.Sci Rep. 2023 Dec 8;13(1):21749. doi: 10.1038/s41598-023-48119-z. Sci Rep. 2023. PMID: 38065963 Free PMC article.
-
Proton transfer during DNA strand separation as a source of mutagenic guanine-cytosine tautomers.Commun Chem. 2022 Nov 5;5(1):144. doi: 10.1038/s42004-022-00760-x. Commun Chem. 2022. PMID: 36697962 Free PMC article.
-
Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.Nucleic Acids Res. 2022 Oct 14;50(18):10601-10613. doi: 10.1093/nar/gkac837. Nucleic Acids Res. 2022. PMID: 36165957 Free PMC article.
-
Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2202489119. doi: 10.1073/pnas.2202489119. Epub 2022 Aug 29. Proc Natl Acad Sci U S A. 2022. PMID: 36037333 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources