A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase.
A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.
Mizuuchi
K
K
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. kmizu@helix.nih.gov
Nobbs
T J
TJ
Halford
S E
SE
Adzuma
K
K
Qin
J
J
D020121:5' Untranslated Regions / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D006868:Hydrolysis; D015183:Restriction Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry; D019895:Transposases / Q000737:chemistry*
D020121:5' Untranslated Regions; D013873:Thionucleotides; D004247:DNA; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; C114697:mu transposase; D019895:Transposases; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases; D017666:Deuterium Oxide