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Kinetics of DNA unwinding by the RecD2 helicase from Deinococcus radiodurans
Author(s) -
Shadrick William R.,
Julin Douglas A.
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.lb37
Subject(s) - deinococcus radiodurans , helicase , dna , biophysics , recbcd , chemistry , processivity , kinetics , enzyme kinetics , homologous recombination , enzyme , biochemistry , dna repair , biology , dna replication , rna , physics , active site , quantum mechanics , gene
RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the E. coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by the D. radiodurans RecD2 under single‐ and multiple‐turnover conditions. There is little unwinding of 20 bp substrates by preformed RecD2‐dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate, using substrates with a 5′‐ssDNA overhang or a forked end. A 12 bp substrate is unwound rapidly under single‐turnover conditions. The unwinding time course can be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant = 5.5 sec 1 , k unw and a dissociation step from partially unwound DNA of k off = 1.9 sec 1 . These results indicate a kinetic step size of about 3 – 4 bp, an unwinding rate of about 15 – 20 bp/sec, and low processivity. The reaction time course data with the 20 bp substrates, determined under multiple‐turnover conditions, can be simulated with a four‐step mechanism and rate constant values very similar to those for the 12 bp substrate. Analysis of reactions done at several RecD2 protein concentrations indicates that the enzyme forms an inactive dimer or other oligomeric form at high enzyme concentrations.