Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1.
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.
Butour
J L
JL
Laboratoire de Pharmacologie et de Toxicologie Fondamentales du CNRS, Toulouse, France.
Mazard
A M
AM
Vieussens
C
C
Johnson
N P
NP
D002945:Cisplatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D006868:Hydrolysis; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D002945:Cisplatin