Article
Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase
Journal of Biological Chemistry
(2006)
Abstract
The T4 helicase loading protein (gp59) interacts with amultitude
of DNA replication proteins. In an effort to determine the functional
consequences of these protein-protein interactions, point
mutations were introduced into the gp59 protein. Mutations were
chosen based on the available crystal structure and focused on
hydrophobic residues with a high degree of solvent accessibility.
Characterization of the mutant proteins revealed a single mutation,
Y122A, which is defective in polymerase binding and has weakened
affinity for the helicase. The interaction between single-stranded
DNA-binding protein and Y122A is unaffected, as is the affinity of
Y122A for DNA substrates. When standard concentrations of helicase
are employed, Y122A is unable to productively load the helicase
onto forked DNA substrates. As a result of the loss of polymerase
binding, Y122A cannot inhibit the polymerase during
nucleotide idling or prevent it from removing the primer strand of a
D-loop. However, Y122A is capable of inhibiting strand displacement
synthesis by polymerase. The retention of strand displacement
inhibition by Y122A, even in the absence of a gp59-polymerase
interaction, indicates that there are two modes of polymerase
inhibition by gp59. Inhibition of the polymerase activity only
requires gp59 to bind to the replication fork, whereas inhibition of
the exonuclease activity requires an interaction between the polymerase
and gp59. The inability of Y122A to interact with both the
polymerase and the helicase suggests a mechanism for polymerase
unlocking by the helicase based on a direct competition between the
helicase and polymerase for an overlapping binding site on gp59.
Keywords
- Crystal structure,
- Hydrophobicity,
- Mutagenesis,
- DNA binding protein,
- glycoprotein gp 41,
- protein y122a,
- single stranded DNA,
- t4 helicase loading protein
Disciplines
Publication Date
January, 2006
DOI
10.1074/jbc.M512185200
Publisher Statement
This article is from Journal of Biological Chemistry 281 (2006): 8697, doi: 10.1074/jbc.M512185200. Posted with permission.
Citation Information
Scott W. Nelson, Jingsong Yang and Stephen J. Benkovic. "Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase" Journal of Biological Chemistry Vol. 281 Iss. 13 (2006) p. 8697 - 8706 Available at: http://works.bepress.com/scott-nelson/5/