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Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication
Author(s) -
Dangerfield Tyler
Publication year - 2019
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.2019.33.1_supplement.776.2
Subject(s) - dna replication , dna polymerase , dna , nucleotide , biophysics , chemistry , polymerase , dna clamp , ternary complex , biochemistry , computational biology , biology , microbiology and biotechnology , enzyme , polymerase chain reaction , gene , reverse transcriptase
The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity. Support or Funding Information The Welch Foundation (F‐1684) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .