In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism

Benedetta A. Sampoli Benítez, Karunesh Arora, Tamar Schlick

Research output: Contribution to journalArticle

Abstract

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase β. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.

Original languageEnglish (US)
Pages (from-to)42-56
Number of pages15
JournalBiophysical Journal
Volume90
Issue number1
DOIs
StatePublished - Jan 2006

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African Swine Fever Virus
Computer Simulation
DNA
Nucleotides
Catalytic Domain
Salts
DNA polymerase X
Thumb
DNA-Directed DNA Polymerase
Molecular Dynamics Simulation
Mutagenesis
DNA Repair
Polymerization
Fingers
Binding Sites
Organizations
Ions

ASJC Scopus subject areas

  • Biophysics

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In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism. / Sampoli Benítez, Benedetta A.; Arora, Karunesh; Schlick, Tamar.

In: Biophysical Journal, Vol. 90, No. 1, 01.2006, p. 42-56.

Research output: Contribution to journalArticle

Sampoli Benítez, Benedetta A. ; Arora, Karunesh ; Schlick, Tamar. / In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism. In: Biophysical Journal. 2006 ; Vol. 90, No. 1. pp. 42-56.
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abstract = "The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase β. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.",
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