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A slow ATP‐induced conformational change limits the rate of DNA binding but not the rate of β clamp binding by the Escherichia coli γ‐complex clamp loader
Author(s) -
Thompson Jennifer Ann,
Paschall Christopher O,
Bloom Linda B
Publication year - 2009
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.23.1_supplement.481.3
Subject(s) - processivity , dna clamp , dna , dna polymerase , biophysics , ternary complex , chemistry , dna replication , atp hydrolysis , biochemistry , biology , enzyme , atpase , rna , reverse transcriptase , gene
In the E. coli DNA replication system, the five‐subunit clamp loader, γ‐complex, loads the βsliding clamp onto DNA. The βclamp, a ring shaped dimer of identical subunits, tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ‐complex binds to βand DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze ATP. Upon ATP hydrolysis, βis released onto DNA and γ‐complex dissociates from the complex. We have developed a βbinding fluorescence assay by covalently attaching a fluorophore, pyrene, onto a Cys residue (Q299C) ofβ. The fluorescence increases two‐fold when γ‐ complex binds β‐pyrene. Using this assay, the rates of association, k on of 3.5 x 10 7 M −1 s −1 and dissociation, k off of 0.005 s −1 of γ‐complex binding to βwere determined in real time. In addition, the kinetics of γ‐ complex binding to DNA were measured in real time to compare to those for βbinding. When γ‐complex is pre‐incubated with ATP, the clamp loader can bind βor DNA with no preferred order. When there is no ATP pre‐incubation, γ‐complex must undergo a slow ATP‐induced conformational change before binding to DNA but does not have to undergo this change when binding toβ. Thus, there may be a kinetic preference for γ‐complex to bind βbefore DNA during the clamp loading reaction cycle. This work was supported by NIH grant GM055596.