William M. Scovell

Cis- and trans-diamminedichloroplatinum(II) binding produces different tertiary structural changes on SV40 DNA

Abstract Cis-diamminedichloroplatinum(II), an active antineoplastic agent, and the inactive trans-isomer, bind to and alter the tertiary structure of supercoiled SV40 DNA. Cis-(NH 3 ) 2 PtCl 2 is more effective in unwinding SV40 DNA than is the trans isomer and can unwind the DNA to the positively supercoiled form. This latter effect was not observed for trans-(NH 3 ) 2 PtCl 2 at levels at least four times greater. Perhaps more significant, cis-(NH 3 ) 2 PtCl 2 reduces the electrophoretic mobility of supercoiled DNA at very short reaction times, an effect that the binding of the trans isomer does not produce until the reaction has proceeded at least 50 times longer. These data suggest that the cis-isomer exhibits a mode of binding different than that of the trans isomer, a mode which is responsible for the more effectively unwinding of the DNA.

cis-Diamminedichloroplatinum(II) selectively cross-links high mobility group proteins 1 and 2 to DNA in micrococcal nuclease accessible regions of chromatin.

The reaction of cis-diamminedichloroplatinum(II) with chicken erythrocyte nuclei produces covalent cross-linking of HMG proteins 1, 2 and E to DNA, in addition to cross-links amongst LMG proteins. This is supported by and consistent with the observations that all cross-links are chemically reversed by NaCN treatment, while only the cross-links involving the HMG proteins 1,2 and E are eliminated after a limited digestion with micrococcal nuclease. Having identified the subset of proteins selectively cross-linked to DNA by the bifunctional cis-(NH3)2PtCl2, a tentative model is proposed for the interactions between DNA and HMG proteins 1 and 2 in bulk chromatin. In addition, possible modes of action for this anti-neoplastic drug are suggested in light of these findings.

Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding

High mobility group protein 1 (HMGB1) interacts with DNA and chromatin to influence the regulation of transcription, DNA repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, ATP-independent manner. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N′ and N″) remain stable and exhibit characteristics distinctly different from the canonical nucleosome. These findings complement previous studies that showed (i) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (ii) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that one aspect of the mechanism of HMGB1 action involves a restructuring of the nucleosome that appears to relax structural constraints within the nucleosome.

Cis-diamminedichloroplatinum(II) modified DNA stimulates far greater levels of S1 nuclease sensitive regions than does the modification produced by the trans- isomer

Abstract S1 endonuclease recognizes distortions in DNA structure produced by both cis- and trans-diamminedichloroplatinum(II) binding. However, cis-(NH 3 ) 2 PtCl 2 binding stimulates far greater levels of S1 digestion than does the trans-isomer. This supports the view that the modes of binding for the two isomers differ and shows that cis-(NH 3 ) 2 PtCl 2 causes a greater disruption of the secondary structure. The S1 digestion products include acid soluble DNA fragments with bound platinum, with the latter providing evidence that (NH 3 ) 2 PtCl 2 is directly responsible for the structural alteration. These findings also reveal that at low levels of binding, the average number of nucleotides excised for each platinum excised in cis-(NH 3 ) 2 PtCl 2 modified DNA is twice as large as for the trans-isomer.

cis-diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle.

The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.

Influence of HMG-1 and Adenovirus Oncoprotein E1A on Early Stages of Transcriptional Preinitiation Complex Assembly

The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription. Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP·TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary HMG-1·TBP·TATA complex, which exhibits increased mobility relative to the TBP·TATA complex. The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB·TBP·TATA complex. However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors. The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.

Expanding the Paradigm for Estrogen Receptor Binding and Transcriptional Activation

Estrogen receptor (ER) binds to a spectrum of functional estrogen response elements (ERE) within the human genome, including ERE half-sites (HERE), inverted and direct repeats. This has been confounding, because ER has been reported to bind weakly, if at all, to these sites in vitro. We show that ER binds strongly to these nonconventional EREs, and the binding is enhanced by the presence of high-mobility group protein B1 (HMGB1). Collectively, these and previous findings reinforce the notion of the plasticity of strong ER/ERE interactions, consistent with their broader range of observed binding specificity. In addition, transient transfection studies using luciferase reporter gene assays show that these EREs drive luciferase activity, and HMGB1 enhances transcriptional activity. Furthermore, HMGB1 gene expression knockdown results in a precipitous drop in luciferase activity, suggesting a prominent role for HMGB1 in activation of estrogen/ER-responsive genes. Therefore, these data advocate that the minimal target site for ER is a cHERE (consensus HERE) that occurs in many different contexts and that HMGB1 enhances both the binding affinity and transcriptional activity. This challenges the current paradigm for ER binding affinity and functional activity and suggests that the paradigm requires significant reevaluation and modification. These findings also suggest a possible mechanism for a cross talk between genes regulated by ER and class II nuclear receptors.

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAs: Influence of (G+C) content and nucleotide sequence

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.

Enhancement of transcription factor, USF, binding to the adenovirus major late promoter: effect of dithiothreitol and high mobility group protein-1

Up-stream stimulatory factor (USF)1 is a human transcription factor which binds specifically to the E-box in the Ad MLP located at - 58 from the start site. The nature of USF binding on a Ad MLP DNA fragment was investigated in the presence of DTT and also in the presence of purified HMG-1 using electrophoretic mobility shift assay. We show that the binding capacity of USF for the E-box increases significantly with increasing DTT concentrations. At the higher DTT levels, a second USF-DNA complex is formed in which there is co-occupation of both the E-box and the initiator sequence. The stability of the second complex is largely refractory to an excess of unlabeled oligonucleotide which contains the initiator sequence. These findings indicate a cooperative binding interaction between USF ligands bound simultaneously at the E-box and the Inr sequence. Two models are proposed which are consistent with these data. Furthermore, experiments indicate that the presence of HMG-1, a nuclear protein known to influence transcriptional activity, increases USF binding activity at the E-box by as much as 100%. These findings indicate that both reducing conditions and HMG-1 may act as modulators of USF-regulated transcription.

Cis-diamminedichloroplatinum(II) modification of SV40 DNA occurs preferentially in (G+C) rich regions: Implications into the mechanism of action

The relative distribution of bound cis- and trans-(NH3)2PtCl2 at specific sites in SV40 DNA is evaluated by monitoring the extent to which five restriction endonucleases, each of which cleave at a single, unique site, are inhibited as a result of the DNA modification. The order of cleavage inhibition is Bgl 1 ≳ Bam HI > Hpa II, Kpn I > Eco RI. Both isomers produce a comparable effect for any particular endonuclease. Inhibition correlates with the % (G+C) content within and about the recognition sequences. That modified sequences immediately adjacent to the recognition sequence influence cleavage is further supported by differential cleavage observed with the multicut Hind III endonuclease. The binding of cis-(NH3)2PtCl2 at the hyper-reactive Bgl 1 site may well be directly responsible for inhibiting SV40 replication.