Biomaterial Database

Stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. 1984

A P Gupta, and S J Benkovic

(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.

UI	MeSH Term	Description	Entries
D008968	Molecular Conformation	The characteristic three-dimensional shape of a molecule.	Molecular Configuration,3D Molecular Structure,Configuration, Molecular,Molecular Structure, Three Dimensional,Three Dimensional Molecular Structure,3D Molecular Structures,Configurations, Molecular,Conformation, Molecular,Conformations, Molecular,Molecular Configurations,Molecular Conformations,Molecular Structure, 3D,Molecular Structures, 3D,Structure, 3D Molecular,Structures, 3D Molecular
D009682	Magnetic Resonance Spectroscopy	Spectroscopic method of measuring the magnetic moment of elementary particles such as atomic nuclei, protons or electrons. It is employed in clinical applications such as NMR Tomography (MAGNETIC RESONANCE IMAGING).	In Vivo NMR Spectroscopy,MR Spectroscopy,Magnetic Resonance,NMR Spectroscopy,NMR Spectroscopy, In Vivo,Nuclear Magnetic Resonance,Spectroscopy, Magnetic Resonance,Spectroscopy, NMR,Spectroscopy, Nuclear Magnetic Resonance,Magnetic Resonance Spectroscopies,Magnetic Resonance, Nuclear,NMR Spectroscopies,Resonance Spectroscopy, Magnetic,Resonance, Magnetic,Resonance, Nuclear Magnetic,Spectroscopies, NMR,Spectroscopy, MR
D011067	Poly dA-dT	Polydeoxyribonucleotides made up of deoxyadenine nucleotides and thymine nucleotides. Present in DNA preparations isolated from crab species. Synthetic preparations have been used extensively in the study of DNA.	Poly(Deoxyadenylate-Thymidylate),Polydeoxyadenine Nucleotides-Polythymine Nucleotides,Poly dA dT,Poly(dA-dT),d(A(5)T(5))2,Nucleotides, Polydeoxyadenine Nucleotides-Polythymine,Nucleotides-Polythymine Nucleotides, Polydeoxyadenine,Polydeoxyadenine Nucleotides Polythymine Nucleotides,dA dT, Poly,dA-dT, Poly,dT, Poly dA
D002621	Chemistry	A basic science concerned with the composition, structure, and properties of matter; and the reactions that occur between substances and the associated energy exchange.
D003838	Deoxyadenine Nucleotides	Adenine nucleotides which contain deoxyribose as the sugar moiety.	Deoxyadenosine Phosphates,Nucleotides, Deoxyadenine,Phosphates, Deoxyadenosine
D004256	DNA Polymerase I	A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair.	DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D005092	Exonucleases	Enzymes that catalyze the release of mononucleotides by the hydrolysis of the terminal bond of deoxyribonucleotide or ribonucleotide chains.	Exonuclease,3'-5'-Exonuclease,3'-5'-Exonucleases,5'-3'-Exonuclease,5'-3'-Exonucleases,3' 5' Exonuclease,3' 5' Exonucleases,5' 3' Exonuclease,5' 3' Exonucleases
D013237	Stereoisomerism	The phenomenon whereby compounds whose molecules have the same number and kind of atoms and the same atomic arrangement, but differ in their spatial relationships. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 5th ed)	Molecular Stereochemistry,Stereoisomers,Stereochemistry, Molecular,Stereoisomer
D013455	Sulfur	An element that is a member of the chalcogen family. It has an atomic symbol S, atomic number 16, and atomic weight [32.059; 32.076]. It is found in the amino acids cysteine and methionine.	Sulfur-16,Sulfur 16
D055598	Chemical Phenomena	The composition, structure, conformation, and properties of atoms and molecules, and their reaction and interaction processes.	Chemical Concepts,Chemical Processes,Physical Chemistry Concepts,Physical Chemistry Processes,Physicochemical Concepts,Physicochemical Phenomena,Physicochemical Processes,Chemical Phenomenon,Chemical Process,Physical Chemistry Phenomena,Physical Chemistry Process,Physicochemical Phenomenon,Physicochemical Process,Chemical Concept,Chemistry Process, Physical,Chemistry Processes, Physical,Concept, Chemical,Concept, Physical Chemistry,Concept, Physicochemical,Concepts, Chemical,Concepts, Physical Chemistry,Concepts, Physicochemical,Phenomena, Chemical,Phenomena, Physical Chemistry,Phenomena, Physicochemical,Phenomenon, Chemical,Phenomenon, Physicochemical,Physical Chemistry Concept,Physicochemical Concept,Process, Chemical,Process, Physical Chemistry,Process, Physicochemical,Processes, Chemical,Processes, Physical Chemistry,Processes, Physicochemical