A novel monofunctional DNA glycosylase activity against thymine glycol in mouse cell nuclei.
Reactive oxygen species continuously oxidize DNA bases and threaten the genetic integrity. Thymine glycol (TG), one of the representative oxidized products, is repaired mainly by base excision repair (BER). In Escherichia coli, endonuclease III (Nth) and endonuclease VIII (Nei) are known to actively remove TG from DNA, and the homologs are well conserved in various organisms. These are bifunctional glycosylases, also associated with apurinic/apyrimidinic (AP) lyase activity. In the present study, a monofunctional TG-DNA glycosylase activity is shown to be one of the predominant nuclear activities present in some mouse tissues. By combining hypertonic extraction and column chromatography, we successfully separated the novel activity from majority of the bifunctional activities. Since it has been reported that mNTH1 may not be a dominant nuclear activity, the monofunctional glycosylase activity, together with mNEIL1, may be the major TG-DNA glycosylases in the mouse nucleus. The optimal reaction conditions for the monofunctional activity were found to be pH 7-8 and 100-150 mM KCl, and the activity was resistant to 20 mM EDTA. High monofunctional activity was detected in the spleen and stomach, while the level was significantly lower in the liver, suggesting that the contribution of the monofunctional activity is variable among organs.
Yamamoto
Ryohei
R
Department of Advanced Pathobiology, Graduate School of Life & Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan. yamamoto@vet.osakafu-u.ac.jp
Akiyama
Miyuki
M
Ide
Hiroshi
H
Yamamoto
Kazuo
K
Matsuyama
Satoshi
S
Kubo
Kihei
K
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002845:Chromatography; D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology; D005470:Fluorometry; D051379:Mice; D008811:Mice, Inbred DBA; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
C029389:thymine glycol; D045647:DNA Glycosylases; D013941:Thymine