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Iso-Migrastatin (iso-MGS) and lactimidomycin (LTM) are glutarimide-containing polyketide natural products (NPs) that are biosynthesized by homologous acyltransferase (AT)-less type I polyketide synthase (PKS) assembly lines. The biological activities of iso-MGS and LTM have inspired numerous efforts to generate analogues via genetic manipulation of their biosynthetic machinery in both native producers and model heterologous hosts. A detailed understanding of the MGS and LTM AT-less type I PKSs would serve to inspire future engineering efforts while advancing the fundamental knowledge of AT-less type I PKS enzymology. The mgs and ltm biosynthetic gene clusters (BGCs) encode for two discrete ATs of the architecture AT-enoylreductase (AT-ER) and AT-type II thioesterase (AT-TE). Herein, we report the functional characterization of the mgsB and ltmB and the mgsH and ltmH gene products, revealing that MgsB and LtmB function as type II thioesterases (TEs) and MgsH and LtmH are the dedicated trans -ATs for the MGS and LTM AT-less type I PKSs. In vivo and in vitro experiments demonstrated that MgsB was devoid of any AT activity, despite the presence of the conserved catalytic triad of canonical ATs. Cross-complementation experiments demonstrated that MgsH and LtmH are functionally interchangeable between the MGS and LTM AT-less type I PKSs. This work sets the stage for future mechanistic studies of AT-less type I PKSs and efforts to engineer the MGS and LTM AT-less type I PKS assembly lines for novel glutarimide-containing polyketides.

The biosynthetic gene cluster for tautomycetin (TTN), a highly potent and selective protein phosphatase (PP) inhibitor isolated from Streptomyces griseochromogenes, has recently been cloned and sequenced. To better understand the transformations responsible for converting the post-polyketide synthase product into the exciting anticancer and immunosuppressive chemotherapeutic candidate TTN, we produced and characterized new analogues resulting from inactivation of two genes, ttnD and ttnF, in S. griseochromogenes. Inactivation of ttnD and ttnF, which encode for putative decarboxylase and dehydratase enzymes, respectively, afforded mutant strains SB13013 and SB13014. The DeltattnD mutant SB13013 accumulated four new TTN analogues, TTN D-1, TTN D-2, TTN D-3, and TTN D-4, whereas the DeltattnF mutant accumulated only one new TTN analogue, TTN F-1. The accumulation of these new TTN analogues defines the function of TtnD and TtnF and the timing of their chemistries in relation to installation of the C5 ketone moiety within TTN. Notably, all new analogues possess a structurally distinguishing carboxylic acid moiety, revealing that TtnD apparently cannot catalyze decarboxylation in the absence of TtnF. Additionally, cytotoxicity and PP inhibition assays reveal the importance of the functional groups installed by TtnDF and, consistent with earlier proposals, the C2''-C5 fragment of TTN to be a critical structural determinant behind the important and unique PP-1 selectivity displayed by TTN.

Charge migration through the DNA base stack has been probed both spectroscopically, to observe the formation of radical intermediates, and biochemically, to assess irreversible oxidative DNA damage. Charge transport and radical trapping were examined in DNA assemblies in the presence of a site-specifically bound methyltransferase HhaI mutant and an intercalating ruthenium photooxidant using the flash-quench technique. The methyltransferase mutant, which can flip out a base and insert a tryptophan side chain within the DNA cavity, is found to activate long-range hole transfer through the base pair stack. Protein-dependent DNA charge transport is observed over 50 A with guanine radicals formed >10(6) s(-1); hole transport through DNA over this distance is not rate-limiting. Given the time scale and distance regime, such protein-dependent DNA charge transport chemistry requires consideration physiologically.

Here the effect on DNA-mediated charge transport of binding by a variety of proteins is examined. DNA assemblies were constructed that contain a tethered rhodium intercalator, as photooxidant, as well as two 5'-GG-3' sites flanking the DNA-binding site for the different proteins. By monitoring the ratio of oxidative damage promoted at the guanine doublet situated distal to the protein-binding site versus that at the proximal site as a function of protein binding, the effects of binding the proteins on DNA-mediated charge transport were determined. Proteins examined included both the wild-type and mutant methyltransferase, M.HhaI, which are base-flipping enzymes, the restriction endonuclease R.PvuII, a TATA-binding protein, which kinks the DNA, and the transcription factor Antennapedia homeodomain protein, which binds DNA through a helix-turn-helix motif. In general, it was observed that yields of long-range oxidative damage correlate with protein-dependent alterations in DNA base stacking. Interactions that disturb the DNA pi-stack inhibit DNA charge transport. Alternatively, interactions that promote no helix distortion but, as a result of tight packing, may rigidify the pi-stack, serve instead to enhance the ability of the DNA base pairs to serve as a conduit for charge transport. Thus, protein binding to DNA modulates long-range charge transport both negatively and positively, depending upon the specific protein/DNA interactions in play. Long-range DNA charge transport and this modulation by protein binding may be important to consider physiologically.

FR66979, a drug closely related to the mitomycin C class of antitumor antibiotics, is shown to covalently cross-link DNA to the DNA-binding domain of the High Mobility Group I/Y (HMG I/Y) DNA-binding proteins in the minor groove.

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.

Abstract The ability of the π-stacked array of heterocyclic DNA bases to behave as an efficient conduit for charge migration has been explored using a wide array of experimental approaches. Spectroscopic studies and biochemical assays show that charge transfer through well-stacked DNA can be extremely facile, although sensitive to structural distortions within the DNA base stack. The efficiency of these long-range reactions depends upon the coupling of the electron donor, acceptor and intervening base pairs within the base stack. As a result, base mismatches and stacking disruptions associated with protein binding to the helix can significantly perturb long range charge transfer. DNA-protein interactions which result in the base flipping of a nucleotide out of the DNA π-stack, in particular, dramatically inhibit long-range charge transfer through DNA. Whether these reactions that can occur over large molecular distances, be applied in sensing DNA damage, and be modulated by DNA-binding proteins, are exploited within the cell remains to be determined.

Background: The class of antitumor antibiotics that includes FR900482 has a very close structural analogy to the mitomycins, one of which, mitomycin C, has been in widespread clinical use for more than 20 years. Like mitomycin C, these antitumor antibiotics are reductively activated in vivo and covalently cross-link DNA as a result of activity of the mitosene moiety generated on reduction. Owing to differences in structure and the attendant mechanistic differences in bioreductive activation between the mitomycins and FR900482, FR900482 does not produce an adventitious superoxide radical anion during reductive activation and thus does not exhibit oxidative strand scission of DNA. It is postulated that the low clinical toxicity of FR900482 relative to mitomycin C is a direct manifestation of the mechanistic differences of bioreductive activation leading to the highly reactive DNA cross-linking mitosenes., Results: Using Fe(II)-EDTA footprinting, we showed that the two natural products FR900482 (1) and dihydro, FR66979 (3), and the semi-synthetically derived triacetate FK973 (2), display remarkable selectivity for 5' deoxy-CG sequences of DNA, and that this selectivity is abolished upon deletion of the exocyclic N2 amine of either participating guanosine residue. In addition, we investigated the mono alkylation abilities of FR66979 with respect to a number of inosine-substituted oligonucleotides and observed that the FR900482 class of compounds were able to give rise to easily separable orientation isomers of their respective cross-links., Conclusions: The FR900482 class of antitumor antibiotics cross-link DNA in a fashion analogous to the mitomycins. The cross-linking reaction yields two orientation isomers which are of vastly different electrophoretic mobility and which also exhibit radically different DNA-protein recognition properties upon reaction with AluI restriction endonuclease. In addition, mono-alkylation of DNA by FR66979 shows little, if any, dependence upon pre-covalent interactions deemed necessary for the mitomycins. These insights support the proposal that the FR900482 class of compounds represents a compelling clinical replacement for mitomycin C, given its greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy.