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Novel inhibition of dATP‐dependent non‐template‐directed nucleotide addition
Author(s) -
Nosé Kirstin Yasuko,
Islas Angel L.
Publication year - 2006
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.20.4.a481
Subject(s) - klenow fragment , dna polymerase , dna polymerase i , exonuclease , nucleotide , dna synthesis , polymerase , microbiology and biotechnology , dna replication , oligonucleotide , chemistry , dna , point mutation , biochemistry , biology , polymerase chain reaction , mutation , gene , reverse transcriptase
DNA polymerases play a crucial role in the replication of an organism’s genome. They are essential in the synthesis of DNA using continuous template strands. In addition to conventional synthesis, however, some DNA polymerases are capable of nucleotide addition without template direction. This non‐template‐directed nucleotide addition (NTA), has many important implications including the possibility for mutations as well as DNA repair. Previous findings point to a significant level of inhibition during NTA when all dNTP’s were present in a reaction, compared to reactions where only dATP was present (Faucett and Islas, Biochem. Biophys. Res. Comm. 337:1030‐1037 [2005]). NTA in the presence of only dATP shows high levels of product formation (190nM after 5 min.) whereas in the presence of all four dNTP’s, the rate of NTA is much reduced (70nM after 5 min.) suggesting that one or more nucleotides is inhibiting ATP‐dependent NTA (approximately 63% inhibition). This study consisted of an in‐depth kinetic analysis of NTA mediated by the 3′‐5′ exonuclease deficient large (Klenow) fragment of Escherichia coli DNA Polymerase I. In order to test the rate of NTA, a Cy‐5 based oligonucleotide assay was used. In vitro experiments were conducted testing each of three dNTP’s against a dATP concentration gradient. Results from these experiments showed that while dCTP did not inhibit, and dTTP showed minimal levels of inhibition, dGTP data exhibited a significant level of competitive inhibition. Results suggest that dGTP can account for almost all of the inhibition seen during non‐template addition in the presence of all dNTP’s. These results have profound significance for understanding the molecular basis of nucleotide recognition by DNA polymerases. This work was supported in part by a grant from the National Science Foundation (NCB – 0315762).