Chemistry of thermal degradation of abasic sites in DNA. Mechanistic investigation on thermal DNA strand cleavage of alkylated DNA.

Abstract

The chemistry of thermal degradation of aldehydic abasic sites in DNA was investigated. Sequencing gel analysis of duocarmycin A-treated 5'-32P-end-labeled DNA fragment indicated that upon heating at neutral pH alkylated DNA was cleaved to provide fragments possessing a modified sugar moiety which is readily decomposed to 3'-phosphate terminus by piperidine treatment. To identify the structure of modified sugar product and to investigate the mechanism of thermal cleavage, thermal degradation of various oligonucleotides containing abasic sites was investigated in detail. It was found that heating the DNA containing an abasic site induces beta-elimination to provide 3'-termini possessing a trans-alpha,beta-unsaturated aldose residue together with 5'-phosphate termini. Upon prolonged heating at pH 7.0, the trans-alpha,beta-unsaturated aldose terminus is isomerized to a cis isomer or is further degraded to its hydrated products and a 3'-phosphate terminus via delta-elimination. This type of thermal degradation also occurs in the abasic site-containing calf thymus DNA. Investigation of the stereochemical course of the thermal beta-elimination reaction using a 2-pro-R-D-containing abasic site has demonstrated that the reaction proceeds via a syn-elimination process as observed for the enzymatic reaction of UV endonuclease V and endonuclease III.