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Residues of Escherichia coli thioredoxin critical for interaction with phage T7 DNA polymerase to increase processivity
Author(s) -
Lee SeungJoo,
Tran Ngoc,
Richardson Charles C
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.592.3
Subject(s) - processivity , dna polymerase , polymerase , dna polymerase i , thioredoxin , biology , dna clamp , escherichia coli , microbiology and biotechnology , dna polymerase ii , biochemistry , dna , gene , polymerase chain reaction , reverse transcriptase
Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to greatly enhance processivity of its DNA polymerase. The structure and amino acid sequence of thioredoxins of all organisms are highly conserved. Therefore, thioredoxins from other organisms may interact with T7 DNA polymerase to support phage growth. This hypothesis was examined using human thioredoxin whose x‐ray crystallographic structure overlaps completely with that of the Escherichia coli protein. However, human thioredoxin cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by isothermal calorimetry and surface plasmon resonance, and thus does not stimulate the polymerase activity. Homologous scanning analysis using this non‐functional homolog reveals that the N‐terminal 20 and the C‐terminal 12 amino acid residues of Escherichia coli thioredoxin can be substituted for its human counterpart without significantly losing ability to support the phage growth. Further investigation using site‐directed mutagenesis identifies leucine‐95 as a critical element that may contribute to hydrophobic interaction with the thioredoxin‐binding loop of the polymerase. The results suggest that specific interactions at the junction of thioredoxin and the DNA polymerase rather than the overall structure are important to enhance the processivity of T7 DNA polymerase.