Biomaterial Database

Mechanism of DNA-dependent protein kinase inhibition by cis-diamminedichloroplatinum(II)-damaged DNA. 1997

J J Turchi, and S M Patrick, and K M Henkels

Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA. jturchi@sirius.wright.edu

We have determined the mechanism of DNA-dependent protein kinase (DNA-PK) inhibition by cis-diamminedichloroplatinum(II)-(cisplatin-) damaged DNA. We previously have demonstrated that Ku, the DNA binding subunit of DNA-PK, is capable of binding to DNA duplexes globally damaged with cisplatin but was unable to stimulate DNA-PKcs, the catalytic subunit [Turchi & Henkels (1996) J. Biol. Chem. 271, 2992-3000]. In this report we have assessed Ku binding and DNA-PK stimulation using a series of DNA substrates containing single, site-specific d(GpG), d(ApG), and d(GpXpG) intrastrand cisplatin adducts and a substrate with a single interstrand cisplatin adduct. Results demonstrate that Ku binding is marginally decreased by the presence of cisplatin adducts on each substrate. When assayed for the ability to stimulate DNA-PK, each cisplatin-damaged substrate resulted in significantly decreased activity compared to undamaged DNA controls. The degree of inhibition of both Ku binding and kinase activity varied depending on the specific adduct employed. The inhibition of DNA-PK activity by cisplatin-damaged DNA was observed using either a synthetic peptide or human replication protein A as a substrate. Autophosphorylation of the DNA-PKcs and Ku subunits was also inhibited in reactions performed with cisplatin-damaged DNA, demonstrating that increased autophosphorylation of DNA-PKcs does not account for the decreased kinase activity observed with cisplatin-damaged DNA. Equilibrium binding and initial velocity experiments revealed a less than 2-fold increase in the Kd of Ku and the Km of DNA-PK for DNA containing a single 1,2-d(GpG) cisplatin adduct. The mechanism of DNA-PK inhibition by cisplatin-damaged DNA can be attributed to a large decrease in the Vmax and small increase in Km.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D009687	Nuclear Proteins	Proteins found in the nucleus of a cell. Do not confuse with NUCLEOPROTEINS which are proteins conjugated with nucleic acids, that are not necessarily present in the nucleus.	Nucleolar Protein,Nucleolar Proteins,Nuclear Protein,Protein, Nuclear,Protein, Nucleolar,Proteins, Nuclear,Proteins, Nucleolar
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D010766	Phosphorylation	The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.	Phosphorylations
D002945	Cisplatin	An inorganic and water-soluble platinum complex. After undergoing hydrolysis, it reacts with DNA to produce both intra and interstrand crosslinks. These crosslinks appear to impair replication and transcription of DNA. The cytotoxicity of cisplatin correlates with cellular arrest in the G2 phase of the cell cycle.	Platinum Diamminodichloride,cis-Diamminedichloroplatinum(II),cis-Dichlorodiammineplatinum(II),Biocisplatinum,Dichlorodiammineplatinum,NSC-119875,Platidiam,Platino,Platinol,cis-Diamminedichloroplatinum,cis-Platinum,Diamminodichloride, Platinum,cis Diamminedichloroplatinum,cis Platinum
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004249	DNA Damage	Injuries to DNA that introduce deviations from its normal, intact structure and which may, if left unrepaired, result in a MUTATION or a block of DNA REPLICATION. These deviations may be caused by physical or chemical agents and occur by natural or unnatural, introduced circumstances. They include the introduction of illegitimate bases during replication or by deamination or other modification of bases; the loss of a base from the DNA backbone leaving an abasic site; single-strand breaks; double strand breaks; and intrastrand (PYRIMIDINE DIMERS) or interstrand crosslinking. Damage can often be repaired (DNA REPAIR). If the damage is extensive, it can induce APOPTOSIS.	DNA Injury,DNA Lesion,DNA Lesions,Genotoxic Stress,Stress, Genotoxic,Injury, DNA,DNA Injuries
D004268	DNA-Binding Proteins	Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.	DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D004791	Enzyme Inhibitors	Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.	Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme