Premium
Molecular Basis of dTTP Specificity by Gene 4 DNA Helicase of Bacteriophage T7
Author(s) -
Lee SeungJoo,
Richardson Charles C
Publication year - 2008
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.22.1_supplement.593.5
Subject(s) - helicase , dna , rna helicase a , biochemistry , gene , nucleotide , amino acid , nucleic acid , bacteriophage , biology , atp hydrolysis , chemistry , enzyme , rna , escherichia coli , atpase
Translocation on ssDNA and the unwinding of dsDNA into ssDNA is mediated by DNA helicases at the expense of hydrolysis of nucleotide triphosphate (NTP). While most of helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To understand the molecular basis of this specificity we have examined the efficiency of unwinding of dsDNA by T7 helicase using a variety of NTPs and their analogs. The 5‐methyl group of thymidine is critical for the efficient unwinding of DNA although the presence of a 3′‐ribosyl hydroxyl group partially overcomes this requirement. In order to identify specific interaction between the NTP and residues within the NTP binding pocket of the protein, several amino acid residues, selected from structural data, were replaced with random amino acid residues. The resulting library of altered gene 4 proteins led to the identification of those residues (T320, R504, Y535, and L542) that are critical for NTP utilization by T7 helicase. Interestingly, some altered proteins containing a single amino acid replacement exhibit NTP utilization profiles distinct from the wild‐type helicase. One of these examples is gp4‐R504G that utilizes dATP more efficiently than dTTP. This altered protein can also utilize dCTP which is barely used by the wild‐type protein. We suggest that the NTP must be stabilized by specific interactions within the NTP binding site of the protein to achieve efficient hydrolysis. These interactions dictate the NTP specificity. This work was supported by Public Health Service Grant GM 54397‐4.