Gamma Ray induced deoxyribonucleic acid strand breaks. 3' Glycolate termini.

L O Rodriguez,W A Haseltine,W D Henner,S M Hecht

doi:10.1016/s0021-9258(18)33104-1

Abstract

Gamma irradiation of DNA in aqueous solution produces single strand breaks with two types of 3'-termini. These termini have been identified as a simple phosphoryl group and a glycolate moiety attached to the 3'-phosphoryl group via the 2"-OH of glycolic acid. Irradiation of 5'-end labeled poly(dG) produces compounds that comigrate with authentic 3',5'-dGDP and 2'-deoxyguanosine-3'-(phospho-2"-O-glycolic acid-5'-monophosphate by thin layer chromatography and polyacrylamide gel electrophoresis. Irradiation of poly(dGdC) followed by digestion to 3'-nucleotides produces 2'-deoxyguanosine-3'-(phospho-2"-O-glycolic acid). The presence of glycolate termini at some strand breaks may affect intracellular repair of these lesions.

Highlights

From the /(Laboratoryof Biochemical Pharmacologya n d +Department of Medical Oncology, Sidney Farber Cancer Institute, Boston, Massachusetts 02115 a n d the YDepartments of Chemistry and Biology, University of Virginia, Charlottesuille, Virginia 22901 end-labeled in a reaction with[y-’”P]ATP (3100 Ci/mmol) and T4 polynucleotide kinase (8)
Preparation of 3‘-dGMP-glycolatel-The desired 3’-dGMP phosphodiester was prepared in analogy with the work of Letsinger et al (9) by the coupling of N2,0v-bis-monomethoxytrityl-2’-deoxyguanosine (0.24 mmol)andthe 2’-O-phosphorodichloriditederivative of y irradiation of DNA in aqueous solution produces single strand breakswith two types of 3”termini. These terminihavebeenidentified as asimplephosphoryl group and a glycolate moiety attached to the 3”phosphoryl groupvia the 2”-OH of glycolic acid
After incubation for 60 min a t 37 “C, 15 units of potato apyrase was added to Breaks in thephosphodiester backbone of DNA are one of degrade unreacted ATP. ‘”P-labeled 3’,5”dGDP and 3’,5”dGDP-glythe major typesof cellular damage induced by ionizing radiation

Summary

Introduction

From the /(Laboratoryof Biochemical Pharmacologya n d +Department of Medical Oncology, Sidney Farber Cancer Institute, Boston, Massachusetts 02115 a n d the YDepartments of Chemistry and Biology, University of Virginia, Charlottesuille, Virginia 22901 end-labeled in a reaction with[y-’”P]ATP (3100 Ci/mmol) and T4 polynucleotide kinase (8). Successive treatments with 80% aqueous acetic acid (25 “C, 8 h) and of 5’-end labeled poly(dG) produces compountdhsat comigrate with authentic 3’,5’-dGDP and 2’-deoxyguanosine-3’-(phospho-2”-O-glycolicacid-5’-monophosphate by thin layer chromatography and polyacrylamidegel electrophoresis. That one of the two major 3’-terminal groups produced by y radiation fragments DNA at each nucleotide, producing ionizing radiation is 2’-deoxynucleoside-3‘-(phospho-2”-O-gly-both phosphoryl and non-phosphoryl group 3’-termini (5).

Results

Conclusion