AT13148 Hedamycin isolated from Streptomyces griseoruber bel
Hedamycin, isolated from Streptomyces griseoruber, belongs to the highly cytotoxic pluramycin class of AT13148 (Figure 3(c)). It consists of a planar anthrapyrantrione chromophore attached to two amino sugar rings at one end and a bisepoxide-containing side-chain at the other end (Figure 3(c)). Hedamycin binds to dsDNA by both reversible and non-reversible modes, and produces a 20 deg. C increase in the DNA melting temperature. Binding to DNA involves an initial reversible intercalation of the chromophore, followed by a much slower covalent binding to the DNA bases. Intercalation into DNA occurs in the major groove by a threading mechanism, and by interactions of the carbohydrate moieties with the major and minor grooves at 5′-NGC sequences, which selectively aligns the epoxide in the major groove for the nucleophilic attack of the N7 of guanine. Studies of the alkylated complexes of hedamycin and altromycin B (another pluramycin antibiotic) with oligonucleotide substrates demonstrate that the glycosidic substituents determine sequence selectivity.58., 59., 60. The modification of DNA by hedamycin results in inhibition of both DNA and RNA polymerases,, and disruption of transcription factor–DNA complexes in vitro.,
To date, two studies evaluating the ability of anti-cancer agents to inhibit RecBCD enzyme have been done. Cisplatin and psoralen were evaluated for their ability to inhibit RecB and RecBCD, respectively. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intra-strand and inter-strand crosslinks, which induce structural distortions in DNA. Cisplatin intra-strand DNA adducts inhibited both the DNA helicase and ATPase activities of RecB. In contrast, psoralen inter-strand cross-links inhibited the helicase activity of RecBCD, but had no effect on the hydrolysis of ATP, which is consistent with an uncoupling of the DNA helicase and ATPase activities of the enzyme.
The goal of this study was to determine and characterize the effects of adozelesin, Et743 and hedamycin on the DNA helicase activity of the RecBCD enzyme. In contrast to previous work with other DNA helicases, and because the substrate for RecBCD is typically DNA molecules greater than several hundred base-pairs in length, we used plasmid-length DNA substrates (i.e. 4100 bp in length). Two complementary DNA helicase assays (fluorescence-based and agarose gel-based) were used to evaluate the effects of each drug on DNA unwinding. The results show that each drug is able to inhibit RecBCD to varying degrees, with Et743 being the most potent inhibitor. In addition to the helicase activity being inhibited, the agarose gel-based assay reveals that both the nuclease activity and the recognition and response to χ are also affected, with the effects being distinct for each agent. Analysis of the effects on the rate of ATP hydrolysis by the translocating RecBCD enzyme revealed that ATP hydrolysis is not uncoupled from DNA unwinding, although for Et743, a twofold decrease in the efficiency of ATP-utilization was observed. These results suggest that the drug-induced structural changes to the DNA, combined with the increased melting temperature of the duplex, caused the rapidly translocating RecBCD enzyme to slow markedly and to push its way through the site(s) of modification. The ability of the enzyme to force its way through the site of damage provides the nuclease active site an increased opportunity to interact with the unwound ssDNA, resulting in increased nuclease activity. Finally, the results reveal a surprising inhibition of the binding of the E. coli ssDNA-binding protein (SSB) to drug-modified and unwound DNA, suggesting that this may be an additional means of disruption of DNA metabolic processes exerted by alkylating agents.
Discussion Two of the agents used, adozelesin and Et743, bind to dsDNA in the minor groove and were the most potent inhibitors of RecBCD, demonstrating that interactions with the minor groove play a critical function during translocation and DNA unwinding by this enzyme. Residues 245 through 254 of the leading domain of RecB are intimately associated with the DNA duplex in the minor groove. As this domain is responsible for pulling the DNA duplex into the holoenzyme (Figure 2(b)), disruption of these contacts delays the progress of dsDNA into the holoenzyme. Et743 was the most potent inhibitor of RecBCD activity used and its interaction with the DNA results in a significant distortion of the DNA structure, so that the minor groove is widened and the DNA is bent by 17(±3)° towards the major groove. Thus, one aspect of the mechanism of inhibition by Et743 is to disrupt the interaction of the protein with duplex DNA ahead of the translocating enzyme and thereby delay the entry of dsDNA into the enzyme. Additional inhibition by Et743 arises due to the increase in duplex stability by 19 deg. C. This stabilization may hinder the ability of RecBCD to split the duplex, further delaying the progress of the DNA into and/or through the enzyme. Finally, intra-strand alkylation by Et743 results in the covalent attachment of an adduct that remains covalently bound to the unwound ssDNA and requires heating to be removed., This adduct is 14–16 Å in size and is as large as the diameter of portions of the channels in RecC that the unwound DNA passes through (11–16 Å in diameter; Figure 2(b)). Thus, the progress of the Et743-adducted, unwound ssDNA through RecC is impeded, resulting in an overall reduction in the rate of DNA translocation. The above-mentioned effects combine to slow the passage of duplex DNA into, and ssDNA through the enzyme, thereby providing the nuclease domain additional time to interact with the unwound ssDNA, resulting in more frequent cleavage of the unwound ssDNA. This is visualized as an increase in the level of a subpopulation of short ssDNA fragments migrating at the lower parts of the gel, and a decrease in the level of full-length unwound ssDNA (Figure 5, Figure 8).