Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen.
Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.
Ko
Rinkei
R
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, 97331-7301, USA.
Bennett
Samuel E
SE
D002384:Catalysis / Q000187:drug effects; D002467:Cell Nucleus; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D045647:DNA Glycosylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008670:Metals / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051818:Replication Protein C / Q000494:pharmacology
D008670:Metals; D018809:Proliferating Cell Nuclear Antigen; C492570:CCNO protein, human; D045647:DNA Glycosylases; D051818:Replication Protein C