BLOCKING OF IN-VITRO DNA-REPLICATION BY DEOXYCYTIDINE ADDUCTS OF THE MUTAGEN AND CLASTOGEN 2-BROMOACROLEIN

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA ), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DN A was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-H-3]dTTP incorporation. M13 mp19 single-stranded DNA templa te modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia c oli DNA polymerase I. It was found that DNA replication stopped freque ntly before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduc t may have been formed in the reaction of 2BA with M13 DNA. To investi gate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by H-1 and C-13 nuclear magnetic resonance and with fast atom bombardmen t mass spectrometry as two diastereomeric ,4-dihydro-2-hydroxy-(2H,7H) [1,6-a]pyrimidin-6-one adducts and a bofuranosyl)-(4H,7H)-pyrimido[1,6 -a]pyrimidin6-one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicit y may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reactive metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discu ssed.