Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): baicalin and genistin.
Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.
Uchiyama
Yukinobu
Y
Department of Applied Biological Science and Frontier Research Center for Genomic Drug Discovery, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Tagami
Junko
J
Kamisuki
Shinji
S
Kasai
Nobuyuki
N
Oshige
Masahiko
M
Chiku
Hiroyuki
H
Ibe
Siroh
S
Koiwai
Osamu
O
Sugawara
Fumio
F
Sakaguchi
Kengo
K
D000818:Animals; D004253:DNA Nucleotidylexotransferase / Q000037:antagonists & inhibitors*; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors; D017931:DNA Primers; D044950:Flavanones / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D007700:Kinetics; D011066:Poly C / Q000494:pharmacology; D051381:Rats; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D019914:Taq Polymerase / Q000037:antagonists & inhibitors; D013722:Teprotide / Q000494:pharmacology*
D017931:DNA Primers; D044950:Flavanones; D005419:Flavonoids; D007529:Isoflavones; C040641:genistin; D012710:Serum Albumin, Bovine; D011066:Poly C; C038044:baicalin; C006680:baicalein; D013722:Teprotide; C415367:DNA polymerase beta2; D019914:Taq Polymerase; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D019951:DNA Polymerase beta