Julia Yaneva

Proteins that specifically recognize cisplatin-damaged DNA: a clue to anticancer activity of cisplatin

Cisplatin, but not its trans geometric isomer, is a potent anticancer drug whose biological activity is a consequence of the formation of covalent adducts between the platinum compound and certain bases in DNA. Two classes of proteins have recently been identified that bind preferentially to damaged sites: proteins that specifically recognize those sites as a first step in their repair, and those that bind to such sites by virtue of structural similarity between the modified DNA and their own natural binding sites. Both classes of proteins may be involved, perhaps in opposing ways, in the cytotoxic effect of the drug.

Interaction of histones H1 and H1° with superhelical and linear DNA

By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1° to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid pαGD containing part of the 5′ flanking and part of the coding sequence of the mouse α‐globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCI and at a histone/DNA ratio of 1.0 and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1° possess a strong binding preference for supercoiled DNA forms. Thus, histone H1° resembles the regular somatic set of histone H1 and not the other differentiation‐specific histone H5 studied thus far.

Linker Histones Do Not Interact with DNA Containing a Single Interstrand Cross-Link Created by Cisplatin

Abstract During our earlier investigations we have observed a prominent preference of the linker histone H1 for binding to a cis-platinated DNA (a synthetic fragment with global type of platination in respect to targets for cisplatin) comparing with unmodified and trans-Pt-modified DNA. In the present work we report our recent experimental results on the binding of the linker histones H1 and H5 to a cisplatin-modified synthetic DNA fragment containing a single nucleotide target d(GC/CG) for inter-platination. Surprisingly, no preferential binding of linker histones to cis-inter-platinated DNA was observed by means of the electromobilityshift assay. The same negative results were obtained with a part of the linker histone molecule suggested to be responsible for DNA-binding - its globular domain. Contrary, the data with another nuclear protein with similar DNA-binding properties as linker histones - HMGB1 - showed a strong afinity for interaction with DNA containing interstrand cross-links.

INTERACTION OF HISTONE H1 WITH CIS-PLATINUM MODIFIED DNA

Abstract Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer frans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.

An indirect-labeling procedure for the study of specific interactions of proteins with DNA.

A semiquantitative and reproducible indirect-labeling procedure for the study of specific protein/DNA interactions using nitrocellulose-filter-immobilized proteins and linear or superhelical DNA molecules is reported. Proteins were immobilized on nitrocellulose filters either by direct dotting or by electrotransfer from polyacrylamide electrophoretic gels. After incubation with the respective DNA (linear restriction fragments or closed circular recombinant DNA plasmids) the paper strips were washed, and specifically bound DNA was denatured by alkali and detected by hybridization with 32P-nick-translated DNA. The quantitation of the reaction was performed by scanning of the autoradiograms of the radioactive spots and determination of the area under the respective peaks. The intensity of the radioactive spots was proportional to the amount of protein present in the dot. The sensitivity of the assay depends primarily on the affinity of the respective DNA to the protein and in the case of mouse liver histone H1AB/mouse alpha-globin gene approximately 50 ng of protein per dot was enough for determination.

Highly Preferential Linker Histone Binding to Actinomycin D-Treated DNA

ABSTRACT Electromobility-shift assay (EMSA) was applied for studying the competition between the anticancer antibiotic actinomycin D (ACTD) and linker histones H1 and H5 for binding to DNA. ACTD is widely used in the treatment of some malignancies. Its binding to DNA occurs in two steps: initial intercalation between guanine and cytosine bases followed by invading the minor groove of the polynucleotide chain. As a consequence of this structural perturbation of DNA, a strong interference with the binding of important regulatory and functional proteins to DNA may occur. Here we present our recent experimental data obtained in a drug-competition assay between ACTD and linker histones H1 and H5 under the following conditions: i) simultaneous incubation of DNA with the antibiotic and histone; ii) preincubation of DNA with ACTD and subsequent addition of the histone; and iii) preincubation of DNA with histone and subsequent addition of the drug. Surprisingly, linker histones bound more effectively to ACTD-pretreated DNA than to the untreated control. The results obtained with plasmid DNA were confirmed using a series of various native and synthetic DNA fragments bearing different number of antibiotic targets. The same results were obtained with the globular domain of H5, the portion of the linker histone suggested to be most responsible for DNA binding. The data with another nuclear protein with similar DNA-binding properties as linker histones—HMGB1—showed strongly reduced contacts with ACTD-treated DNA compared with the untreated.

The electrophoretic separation of curved cisplatin-modified DNA fragments on polyacrylamide gels is dependent on the voltage gradient.

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The param eters of the electric field do not affect separation of ‘normal’ DNA fragments of comparable length.