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J Mol Biol. 2010 Jul 16;400(3):295-308. doi: 10.1016/j.jmb.2010.05.030. Epub 2010 May 21.

Identification of a new motif in family B DNA polymerases by mutational analyses of the bacteriophage t4 DNA polymerase.

Author information

1
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.

Abstract

Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low-dGTP conditions either by correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype-sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low-dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs.

PMID:
20493878
PMCID:
PMC3153374
DOI:
10.1016/j.jmb.2010.05.030
[Indexed for MEDLINE]
Free PMC Article

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