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Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1
J L Butour1, A M Mazard, C Vieussens
1Laboratoire de Pharmacologie et de Toxicologie Fondamentales du CNRS, Toulouse, France.
Abstract:
The antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP) reacts covalently with DNA and disrupts its secondary structure. Damaged DNA, but not native DNA, is readily digested by S1 nuclease, an endonuclease specific for single stranded polynucleotides. We have measured S1 nuclease digestion of platinated DNA by the release of platinum-DNA adducts and compared it with digestion of unplatinated DNA. The rate of hydrolysis of damaged substrate from platinum-DNA complexes was less than the overall rate of digestion of nucleotides. Similar results were observed for platinum-DNA complexes in native, denatured or renatured conformations. The hydrolysis of denatured platinum-DNA complexes, rb = 0.075 platinum per nucleotide, obeyed Michaelis-Menten kinetics. Taking into account the level of DNA damage, Vm, for the release of platinated adducts was 0.6 times smaller than for digestion of unplatinated DNA. Km values and competition experiments indicated that the enzyme bound equally well to platinated and unplatinated substrates. Similar results were obtained for denatured DNA complexes with trans-DDP while [PtCl(diethylenetriamine)]Cl had no influence on nuclease digestion. These results suggest that bifunctional platinum-DNA lesions have contradictory effects on the hydrolysis of double stranded DNA by S1 nuclease. On one hand they create nuclease sensitive substrate by disrupting DNA secondary structure. On the other, they inhibit digestion of the damaged strand by increasing the activation energy for hydrolysis.
Insights
Cisplatin (cis-DDP) damages DNA, making it susceptible to S1 nuclease digestion. However, platinum-DNA adducts inhibit the enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cis-diamminedichloroplatinum(II) (cis-DDP) is an effective antitumor agent.
- Cis-DDP covalently binds to DNA, altering its structure.
- S1 nuclease specifically digests single-stranded or damaged DNA.
Purpose of the Study:
- To investigate the effect of platinum-DNA adducts on S1 nuclease digestion.
- To compare the digestion rates of platinated and unplatinated DNA.
- To understand the mechanism by which platinum compounds affect DNA hydrolysis.
Main Methods:
- Measuring S1 nuclease digestion of platinated DNA by monitoring platinum-DNA adduct release.
- Comparing digestion rates of native, denatured, and renatured DNA complexes.
- Applying Michaelis-Menten kinetics to analyze enzyme-substrate interactions.
- Conducting competition experiments to assess enzyme binding affinity.
Main Results:
- Platinum-DNA adducts were released slower than unplatinated nucleotides during S1 nuclease digestion.
- S1 nuclease exhibited Michaelis-Menten kinetics with denatured platinum-DNA complexes.
- Enzyme binding (Km) was similar for platinated and unplatinated DNA.
- Bifunctional platinum lesions inhibited the hydrolysis of the damaged DNA strand.
Conclusions:
- Bifunctional platinum-DNA lesions have opposing effects on S1 nuclease activity.
- Lesions create nuclease-sensitive sites by disrupting DNA secondary structure.
- Lesions also inhibit DNA strand hydrolysis by increasing activation energy.