Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

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 paper, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the analysis of model DNA substrates acted upon 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 β-elimination product as previously described. Upon the basis of 18O 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 upon 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 upon these lesions may influence the biological outcome of damage repair.

An HPLC–tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products

“…AP sites generated by the glycosylase are nicked by alkali treatment or, in the case of the bifunctional enzymes, by intrinsic lyase activity, and the substrate and product fragments separated electrophoretically to quantify the ratio of substrate/product. Fragments have also been analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [29]. The oligonucleotide-based glycosylase assay enables precise characterization of binding and catalysis of a specific lesion under well-defined conditions and nucleotide sequence contexts, and takes advantage of automated chemical DNA synthesis technology and availability of a large number of commercially available phosphoramidite precursors.…”

“…Mass spectrometry approaches have been used to identify modified nucleoside metabolites from rat liver [45], establish the spectrum of lesions produced in genomic DNA by oxidizing [46, 47] and alkylating agents [48-51], and study oxidative damage repair [29, 47, 52-54]. To our knowledge, HPLC-MS/MS techniques capable of simultaneously detecting alkylated DNA adducts present in DNA [44, 48, 50, 51] have not been utilized to assay the excision activity of DNA glycosylases.…”

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

“…3A, lane 8). The latter is unstable and typically exists in the hydrated form (PUA H ) (30,31). These cleavage fragments were therefore identified as the 5Ј-CCACCAAC-p-PUA H sequence, which was confirmed by MALDI-TOF/MS methods (see below).…”

“…3A, lane 8). In this fragment the PUA H is the hydrated form (-CH 2 CHOHCHOHCH 2 CHO, M ϭ 117.0 Da) of the aldehyde since its mass is 18 Da higher than the mass of the ␣,␤-unsaturated aldehyde (-CH 2 CHOHCH ϭ CHCHO, M ϭ 99.0 Da) (30,31). Again the G* base is missing from all of these oligonucleotide fragments; furthermore the former T* base is present in its intact form T in all 3Ј-downstream cleavage fragments.…”

An electrochemiluminescence sensing for DNA glycosylase assay with enhanced host-guest recognition technique based on α-cyclodextrin functionalized gold/silica cell-shell nanoparticles

“…The most common methods to assay the activity of hOGG 1 are 32 P-labeled fragments using gel electrophoresis [10], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [11], and liquid chromatography-mass spectrometric (LC-MS) [12]. These methods indirectly investigate the activity of hOGG 1 by quantifying the amount of the released base or the amount of AP site strand.…”