Abstract
Dehydroaltenusin is a selective inhibitor of mammalian DNA polymerase α (pol α) from a fungus (Alternaria tennuis). We have designed, synthesized, and characterized a derivative of dehydroaltenusin conjugated with a C12-alkyl side chain (dehydroaltenusin-C12 [C12]). C12 was the strongest pol α inhibitor in vitro. We introduced C12 into NIH3T3 cells with the help of a hypotonic shift, that is, a transient exposure of cultured cells in hypotonic buffer with small molecules which can not penetrate cells. The cells that took in C12 by hypotonic shift showed cell growth inhibition. At a low concentration (5 μM), DNA replication was inhibited and several large replication protein A (RPA) foci, which is different from dUTP foci. Furthermore, when C12 was incubated with aphidicolin, RPA foci were not observed in cells. Finally, these findings suggest that C12 inhibited DNA replication through pol α inhibition, and generated single-stranded DNA, resulted in uncoupling of the leading strand and lagging strand synthesis. These findings suggest that C12 could be more interesting as a molecule probe or anticancer agent than aphidicolin. C12 might provide novel markers for the development of antiproliferative drugs.
Highlights
Metazoan organisms are known to contain at least 14 DNA polymerases [1]
We reported that a longer fatty acid chain is a stronger inhibitor than a short fatty acid chain [2]; the solubility of the longer fatty acid chain is poor in aqueous media
We have designed and chemically synthesized a potent inhibitor, C12, which is a derivative of dehydroaltenusin conjugated with a C12alkyl group, to analyze the details of DNA replication in mammalian cells
Summary
Metazoan organisms are known to contain at least 14 DNA polymerases [1]. we have screened for natural compounds that were selective inhibitors of these polymerases [2,3,4].Selective inhibitors of DNA polymerases must be useful tools in distinguishing DNA polymerases and clarifying their biological and in vivo functions [1, 5]. Polymerase [E.C. 2.7.7.7], pol) α; Enzyme inhibitor; DNA replication; Replication fork uncoupling; Hypotonic shift; Molecule probe; Anticancer drug. Polymerase inhibitor aphidicolin demonstrated that DNA polymerases α, δ and ε (pol α, δ and ε) are essential for replication [6]. We chemically synthesized a derivative of dehydroaltenusin, which is stronger pol α inhibitor than dehydroaltenusin, and this compound was conjugated with a C12-saturated alkyl group (i.e., lauric acid) (C12, 2, Figure 1).
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