Specific association of cyclin-like uracil-DNA glycosylase with the proliferating cell nuclear antigen.
We have previously isolated a human gene that encodes a cyclin-like protein with uracil-removing activity (UDG2) (Muller, S.J., and Caradonna, S. 1993. J. Biol. Chem. 268, 1310-1319). The structural and regulatory similarities shared between this uracil-DNA glycosylase and cyclins suggested that it may interact with additional proteins. Using a unique affinity purification protocol (Ugi-Sepharose) and anti-UDG2 antibodies, we have identified a physical interaction between the cyclin-like uracil-DNA glycosylase and PCNA in extracts derived from HeLa cells. Conversely, we show that anti-PCNA immunoprecipitates possess significant uracil-DNA glycosylase activity. This activity is specifically blocked by the addition of uracil-DNA glycosylase inhibitor protein (Ugi) derived from bacteriophage PBS2. To further characterize this association, we performed in vitro mixing experiments using 35S-labeled PCNA and uracil-DNA glycosylase (UDG2) that were generated in a coupled transcription/translation system. We show that UDG2 and PCNA are coprecipitated using anti-PCNA antibodies and anti-UDG2 antibodies as well as Ugi-Sepharose. When PCNA is preincubated with synthetic peptides corresponding to amino acid residues 73-90 of UDG2, the PCNA-UDG2 association is prevented. By contrast, addition of synthetic peptides corresponding to amino acid residues 208-223 has no effect on this interaction. These findings suggest that the UDG2 domain encompassing amino acids 73-90 is directly involved in binding PCNA.
Muller-Weeks
S J
SJ
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford 08084, USA. muller@umdnj.edu
Caradonna
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D016213:Cyclins; D045647:DNA Glycosylases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000494:pharmacology
D016213:Cyclins; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D018809:Proliferating Cell Nuclear Antigen; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase