Format

Send to

Choose Destination
J Biol Chem. 2004 Jun 18;279(25):26005-12. Epub 2004 Apr 14.

The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity.

Author information

1
Department of Biochemistry, The Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Abstract

Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations. Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA. Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes. Thus, increasing the ssDNA tail length increases the unwinding efficiency. The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules. The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site. The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity. The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated. The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.

PMID:
15087464
DOI:
10.1074/jbc.M403257200
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center