Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

Abstract

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase ␣ (pol ␣) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol ␣. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as ␦ and ⑀; it also showed no effect even on the pol ␣ activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol ␣ was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 M. Dehydroaltenusin-induced inhibition of mammalian pol ␣ activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol ␣, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol ␣. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol ␣, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.Recent investigations have revealed that eukaryotic cells contain at least eight types of DNA polymerase (pol ␣, ␤, ␥, ␦, ⑀, , , and ) (1-4). We screened for natural compounds that selectively inhibit one of the eukaryotic DNA polymerases and found several inhibitors (5-8). Selective inhibitors of DNA polymerases are useful tools and molecular probes to distinguish DNA polymerases and clarify their biological and in vivo functions (9). For example, aphidicolin is a selective inhibitor of both DNA polymerase (pol) 1 ␣ and eukaryotic DNA replicative polymerase, indicating that this polymerase is essential for DNA replication (10). Aphidicolin inhibitor has been very useful for studying the DNA replication system (11); however, there have been no previous reports of inhibitors capable of distinguishing among pol ␣, ␦, and ⑀.We found an interesting inhibitor that influenced only the activity of mammalian pol ␣. The agent, which was determined to be dehydroaltenusin, has been reported to be the inhibitor of myosin light chain kinase (12, 13). Dehydroaltenusin did not affect the activities of replicative DNA polymerases, such as ␦ and ⑀, from calf thymus and HeLa cells, respectively; it also showed no effect on pol ␣ activity even from another vertebrate, i.e. fish, although the amino acid sequences of both DNA polymerases are highly homologous. The agent must be able to determine small structural differences between mammalian and fish pol ␣. Therefore, dehydroaltenusin could be a useful tool with which to study the biochemical functions of pol ␣ and a molecular probe to distinguish the structure of pol ␣. EXPERIMENTAL PROCEDURESMaterials-Nucleotides and chemically synthesized template-primers, such as poly(dA) and oligo(dT) 12-18 , were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). [ 3 H]dTTP (43 Ci/mmol) was pur...

Natural Dibenzo-α-Pyrones and Their Bioactivities

“…This compound also exhibited strong inhibitory activity on mammalian DNA polymerase α in vitro [79]. It was further proved to abrogate cell proliferation of the cultured mammalian cells to show its potential as an effective chemotherapeutic agent against tumors [44].…”

Galactosyldiacylglycerol, a Mammalian DNA Polymerase .ALPHA.-Specific Inhibitor from a Sea Alga, Petalonia bingbamiae.

“…7,8) For DNA polymerases, poly(dA)/oligo(dT) [12][13][14][15][16][17][18] and dTTP were used as the template-primer DNA and nucleotide substrate, respectively. For HIV reverse transcriptase, poly(rA)/ oligo(dT) [12][13][14][15][16][17][18] and dTTP were used as template-primer and nucleotide substrate, respectively. For terminal deoxynucleotidyl transcriptase, oligo(dT) [12][13][14][15][16][17][18] (3Ј-OH) and dTTP were used as template-primer and nucleotide substrate, respectively.…”

“…Nucleotides and chemically synthesized template-primers such as poly(dA) and oligo(dT) [12][13][14][15][16][17][18] were purchased from Pharmacia (Uppsala, Sweden). [ 3 H]-dTTP (43 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Boston, MA, U.S.A.).…”

“…5,6) We screened for natural compounds that selectively inhibit one of many types of DNA polymerase, and found several such inhibitors. [7][8][9][10][11][12][13][14][15][16][17] Selective inhibitors of DNA polymerases will be useful tools to distinguish DNA polymerases and to clarify their in vivo biological functions. 18) For example, aphidicolin is known as a selective inhibitor of eukaryotic DNA replicative polymerases such as a, d and e (pol.…”

“…a species. 17) Inhibitors capable of distinguishing among pol. a, d and e from broad eukaryotic species are required for studies of the DNA replication system.…”