Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism
Open Access
- 17 July 2000
- journal article
- research article
- Published by Springer Nature in The EMBO Journal
- Vol. 19 (14), 3799-3810
- https://doi.org/10.1093/emboj/19.14.3799
Abstract
DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3′‐tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non‐hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single‐stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an ‘active’ mechanism for PcrA that involves two distinct ATP‐dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.Keywords
This publication has 27 references indexed in Scilit:
- DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicaseJournal of Molecular Biology, 1999
- Mechanism of Inorganic Phosphate Interaction with Phosphate Binding Protein fromEscherichia coliBiochemistry, 1998
- PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling‐circle replicationMolecular Microbiology, 1998
- Helicases: a unifying structural theme?Current Opinion in Structural Biology, 1998
- Crystal structure of a DExx box DNA helicaseNature, 1996
- Direct, Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPaseBiochemistry, 1994
- Escherichia coli Rep helicase unwinds DNA by an active mechanismBiochemistry, 1993
- Helicases: amino acid sequence comparisons and structure-function relationshipsCurrent Opinion in Structural Biology, 1993
- Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymeraseBiochemical and Biophysical Research Communications, 1990
- Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)Biochemistry, 1988