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S. cerevisiae RFC Walker A and arginine finger mutants are defective in clamp opening and binding
Author(s) -
Marzahn Melissa Rose,
Bloom Linda B.
Publication year - 2012
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.26.1_supplement.739.8
Subject(s) - walker motifs , mutant , atp hydrolysis , dna replication , atpase , processivity , arginine , dna clamp , biology , proliferating cell nuclear antigen , microbiology and biotechnology , biochemistry , dna , dna polymerase , enzyme , amino acid , rna , reverse transcriptase , gene
Though eukaryotic replication has been studied for over 30 years the exact mechanisms for each enzyme involved are largely unknown. The goal of this study is to kinetically evaluate the function of the clamp and clamp loader. The eukaryotic clamp, proliferating cell nuclear antigen (PCNA), tethers the polymerase to the template DNA and is loaded onto DNA by replication factor C (RFC), a heteropentamer complex belonging to the AAA+ family of ATPases. Several conserved ATPase motifs, the Walker A and B loops and arginine finger motif, coordinate ATP binding and hydrolysis. The Walker A loop participates in ATP binding while the arginine finger motif is involved in sensing bound ATP. Mutation of the Walker A Lys or arginine finger Arg residue dramatically reduces ATP binding or recognition of bound ATP, respectively. Conserved Lys or Arg residues were mutated in each of the 4 ATP sites, and RFC mutants containing a single mutation were characterized to determine the functional role each motif plays. Fluorescence‐based assays were used to determine how these mutations affect RFC's ability to bind DNA and bind and open PCNA in the steady‐state and pre‐steady‐state. Mutations at all sites except one, Rfc4 Arg, caused a dramatic loss of clamp binding and opening. This suggests that ATP binding is essential for clamp binding and opening but that recognition of bound ATP may be an ordered process within the clamp loading reaction.