AP site structural determinants for Fpg specific recognition

Abstract : 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.
Document type :
Journal articles
Complete list of metadatas

https://hal.archives-ouvertes.fr/hal-02141144
Contributor : Laëtitia Legoupil <>
Submitted on : Monday, May 27, 2019 - 4:52:39 PM
Last modification on : Wednesday, May 29, 2019 - 1:17:26 AM

Links full text

Identifiers

Collections

Citation

Bertrand Castaing, Jean-Louis Fourrey, Nadege Hervouet-Coste, Martial Thomas, Serge Boiteux, et al.. AP site structural determinants for Fpg specific recognition. Nucleic Acids Research, Oxford University Press, 1999, 27 (2), pp.608-615. ⟨10.1093/nar/27.2.608⟩. ⟨hal-02141144⟩

Share

Metrics

Record views

26