Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches.
We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.
Nicolas
E
E
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Beggs
J M
JM
Taraschi
T F
TF
D000225:Adenine / Q000378:metabolism*; D020137:Base Pair Mismatch; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D054789:Ribosome Inactivating Proteins, Type 1; D013997:Time Factors; D015032:Zinc / Q000378:metabolism
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D008623:Mercaptoethanol; C025674:GEL protein, Gelonium multiflorum; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D015032:Zinc; D000225:Adenine