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Developing Reporter Systems to Monitor the Structural Dynamics of MutS
Author(s) -
Doucette Christopher William,
Biro F. Noah
Publication year - 2010
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.24.1_supplement.876.2
Subject(s) - dna mismatch repair , dna , base pair , msh2 , chemistry , biophysics , msh6 , dna repair , biology , microbiology and biotechnology , biochemistry
DNA mismatch repair (MMR) is an evolutionarily conserved process that locates and repairs post‐replicative errors in nascent DNA and is critical for maintaining the genome integrity. The first step in the MMR pathway is recognition of the base‐base mismatch/insertion deletion loop (IDL) by MutS in prokaryotes and the M ut S h omologues, Msh2‐Msh6 and Msh2‐Msh3, in eukaryotes. While there is a significant body of literature concerning DNA‐binding and ATPase activities of MutS, information on MutS conformational changes involved in recognizing an array of non‐Watson‐Crick DNA structures is lacking. Here we present two fluorescence reporter assays developed to examine T. aquaticus MutS conformational changes and better understand the relationship between its structure/dynamics and function. Two mutant proteins were prepared: 1) F39, which contacts DNA, was replaced with tryptophan to monitor intrinsic protein fluorescence during the reaction; 2) M88 in the DNA binding domain was replaced by cysteine and labeled with a thiol reactive fluorophore that is sensitive to protein conformation. We are currently measuring changes in T. aquaticus MutS conformation on mismatch/IDL binding in equilibrium and in real time by transient kinetic analysis.