ABSTRACT
When present in DNA, 3,N(4)-ethenocytosine (epsilon C) residues are potentially mutagenic and carcinogenic in vivo. The enzymatic activity responsible for the repair of the epsilon C residues in human cells is the hTDG protein, the human thymine-DNA-glycosylase that removes thymine in a T/G base pair [Proc. Natl. Acad. Sci., U.S.A., 95 (1998) 8508]. One of the distinctive properties of the hTDG protein is that it remains tightly bound to the AP-site resulting from its glycosylase activity. In this paper we report that the human AP endonuclease, the HAP1 (Ape1, APEX Ref-1) protein, stimulates the processing of epsilon C residues by the hTDG protein in vitro, in a dose-dependent manner. This property of HAP1 protein is specific since E.coli Fpg, Nfo and Nth proteins, all endowed with an AP nicking activity, do not show similar features. The results suggest that the HAP1 protein displaces the hTDG protein bound to the AP-site and therefore increases the turnover of the hTDG protein. However, using a variety of techniques including gel retardation assay, surface plasmon resonance and two-hybrid system, it was not possible to detect evidence for a complex including the substrate, the hTDG and HAP1 proteins.
Subject(s)
Carbon-Oxygen Lyases/metabolism , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA Repair/physiology , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Binding Sites/drug effects , Binding Sites/physiology , Carbon-Oxygen Lyases/chemistry , Carbon-Oxygen Lyases/pharmacology , Cytosine/chemistry , DNA Ligases/chemistry , DNA Ligases/metabolism , DNA Repair/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Formamidopyrimidine Glycosylase , Deoxyribonuclease (Pyrimidine Dimer) , Deoxyribonuclease IV (Phage T4-Induced) , Dose-Response Relationship, Drug , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/pharmacology , Enzyme Activation/drug effects , Humans , Magnesium/pharmacology , N-Glycosyl Hydrolases/metabolism , N-Glycosyl Hydrolases/pharmacology , Oligonucleotides/chemistry , Oligonucleotides/genetics , Oligonucleotides/metabolism , Protein Binding/physiology , Surface Plasmon Resonance , Two-Hybrid System TechniquesABSTRACT
Various forms of oxidative stress lead to the formation of damaged bases including N-(2-deoxy-beta-D-erythro-pentofuranosyl)-N-3-(2R-hydroxyisobutyric acid)-urea or alphaRT, the fragmentation product of thymine formed from 5R-thymidine C5-hydrate upon hydrolysis. It was shown that alphaRT is excised by Escherichia coli Fpg and Nth proteins. Here we report that when present in DNA, alphaRT is, in addition, a substrate for the E. coli AlkA protein with an apparent K(m) value of congruent with170 nM. alphaRT positioned opposite T, dG, dC, and dA were efficiently excised by AlkA protein from duplex oligodeoxynucleotides in the following order: dA approximately T >> dC approximately dG. This is the first example of the excision of a ring opened form of a pyrimidine by AlkA protein and also the first example where the same DNA base lesion is excised by three different DNA glycosylases of the base excision repair pathway. The present results suggest possible structural similarity of the active site between E. coli AlkA, Fpg, and Nth proteins.
