Nucleic Acids Research, Vol 27, Issue 2 608-615, Copyright © 1999 by Oxford University Press
B Castaing, JL Fourrey, N Hervouet, M Thomas, S Boiteux and C Zelwer
The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase
(Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP)
site analogs was investigated by electrophoretic mobility shift assay
(EMSA) and by footprinting experiments. We showed that the reduced AP site
is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp =
0.26 and 0.5 nM, respectively) as compared with the other analogs tested in
this study ( K Dapp >2.8 nM). The 1,3- propanediol (Pr)
residue-containing DNA seems to be the minimal AP site structure allowing a
Fpg specific DNA binding, since the ethyleneglycol residue is not
specifically bound by these enzymes. The newly described cyclopentanol
residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K
Dapp = 2.9 and 25 nM, respectively). These results suggest that the
hemiacetal form of the AP site is negatively discriminated by the Fpg
protein suggesting a hydrogen bond between the C4'-hydroxyl group of the
sugar and a Fpg residue. High-resolution hydroxyl radical footprinting
using a duplex containing Pr shows that Fpg binds to six nucleotides on the
strand containing the AP site and only the base opposite the lesion on the
undamaged complementary strand. This comparative study provides new
information about the molecular mechanism involved in the Fpg AP lyase
activity.
ARTICLES
AP site structural determinants for Fpg specific recognition
Centre de Biophysique Moleculaire, UPR 4301 affiliated to the University of Orleans, CNRS, rue Charles Sadron, 45071 Orleans Cedex 2, France. castaing@cnrs-orleans.fr
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