Stereochemical Course of the 3'→5′-Exonuclease Activity of DNA Polymerase I

Amar P. Gupta, Stephen Benkovic

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

(SP)-2/-Deoxyadenosine 5′-0-[1- 0,1- 0,1,2-O] 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 α-phos-phorus and 12% participation by the β-phosphate to form a cyclic α,β-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the β-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 deoxy-nucleoside 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.

Original languageEnglish (US)
Pages (from-to)5874-5881
Number of pages8
JournalBiochemistry
Volume23
Issue number24
DOIs
StatePublished - Jan 1 1984

All Science Journal Classification (ASJC) codes

  • Biochemistry

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