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 D NA glycosylase activity that removes adenine from single-stranded DNA (Nico las, E,, Beggs, J, M,, Haltiwanger, B, M,, and Taraschi, T. F, (1998) J. Bi ol, Chem. 273, 17216-17220), In this investigation, we further characterize d the interaction of the RIP gelonin with single stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physi ological pH, zinc and p-mercaptoethanol stimulated the adenine DNA glycosyl ase activity of gelonin, Under these conditions, gelonin catalytically remo ved adenine from single-stranded DNA and, albeit to a lesser extent, from n ormal base pairs and mismatches in duplex DNA, Also unprecedented was the f inding that activity on single-stranded and double-stranded oligonucleotide s containing multiple adenines generated unstable products with several aba sic sites, producing strand breakage and duplex melting respectively. The r esults from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A reexamination of the classification of gelonin as a DNA glycosylase/AP l yase 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 mul tisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA m ay be physiologically relevant.