Evaluation of Type-A Endonucleases for the Quantitative Analysis of DNA Damage due to Exposure to Acetaldehyde Using Capillary Electrophoresis

Abstract

The substrate selectivities of three endonucleases were studied quantitatively using capillary zone electrophoresis to find one giving N²-ethyl(Et)-2′-deoxyguanosine-5′-monophosphate (5′-dGMP) and cyclic 1,N²-propano(CPr)-5′-dGMP from DNAs damaged by acetaldehyde (AA). Six 2′-deoxyribonucleoside-5′-monophosphates to be quantified in the hydrolysis solutions of DNAs, namely, Et-5′-dGMP, CPr-5′-dGMP, and four authentic ones, were completely separated using a 100 mM borate running buffer solution having an optimized pH of 9.67. Using the present method, nuclease reactions of nuclease S1 (NS1), nuclease P1 (NP1), and nuclease Bal 31 to 2′-deoxyribonucleoside-5′-monophosphates from damaged Calf thymus (CT-) DNAs were monitored. The CT-DNAs were prepared by treatment with AA to generate Et-guanine or CPr-guanine internally. Bal 31 hydrolyzed the damaged CT-DNAs to yield Et-5′-dGMP and CPr-5′-dGMP quantitatively. The two 5′-dGMP adducts were not detected in the hydrolysis solutions using NS1 or NP1. Bal 31 can be a suitable nuclease for analyzing DNA damages caused by AA.