Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3' and 5' ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple beta-elimination product as described previously. On the basis of (18)O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.
Darwanto
Agus
A
Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA.
Farrel
Alvin
A
Rogstad
Daniel K
DK
Sowers
Lawrence C
LC
D001483:Base Sequence; D004249:DNA Damage; D045647:DNA Glycosylases / Q000032:analysis / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase