Abstract
Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOgg1) initiate the base excision repair pathway for 7,8-dihydro-8-oxoguanine (8-oxoG) residues present in DNA. Recent structural and biochemical studies of Fpg-DNA and hOgg1-DNA complexes point to the existence of extensive interactions between phosphate groups and amino acids. However, the role of these contacts and their physiological relevance remains unclear. In the present study, we combined chemical cross-linking and electrospray ionization mass spectrometry (ESI/MS/MS) approaches to identify interacting residues in the Fpg-DNA and hOgg1-DNA complexes. The active centers of Fpg and hOgg1 were cross-linked with a series of reactive oligonucleotide duplexes containing both a single 8-oxoG residue and an O-ethyl-substituted pyrophosphate internucleotide (SPI) group at different positions in duplex DNA. The cross-linking efficiency reached 50% for Fpg and 30% for hOgg1. We have identified seven phosphate groups on both strands of the DNA duplex specifically interacting with nucleophilic amino acids in Fpg, and eight in hOgg1. MS/MS analysis of the purified proteolytic fragments suggests that lysine 56 of Fpg and lysine 249 of hOgg1 cross-link to the phosphate located 3' to the 8-oxoG residue. Site-specific mutagenesis analysis of Fpg binding to DNA substrate confirms the conclusions of our approach. Our results are consistent with crystallographic data on the Fpg-DNA complex and provide new data on the hOgg1-DNA interaction. The approach developed in this work provides a useful tool to study pro- and eukaryotic homologues of Fpg as well as other repair enzymes.
Highlights
OCTOBER 27, 2006 VOLUME 281 NUMBER 43 glycosylase are DNA repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (8-oxoG) residues from DNA [1,2,3]. 8-OxoG is the major mutagenic base damage generated in DNA by reactive oxygen species produced in aerobic respiration and after cell injury or exposure to physical and chemical oxygen radical-forming agents [4]. 8-OxoG is a miscoding lesion, because it pairs preferentially with adenine instead of cytosine and induces G1⁄7C 3 T1⁄7A transversions in vivo and in vitro [5, 6]
We have identified seven phosphate groups on both strands of the DNA duplex interacting with nucleophilic amino acids in formamidopyrimidine-DNA glycosylase (Fpg), and eight in hOgg1
We present two alternative strategies combining cross-linking and ESI/mass spectrometers to fragment parent ions (MS/MS) to identify the amino acids of 8-oxoguanine-DNA glycosylases interacting with DNA phosphate groups under physiological conditions
Summary
Isolation of Oligonucleotide-Fpg Peptide Conjugate—After preparative cross-linking, the reaction mixture was denatured in 0.1% SDS at 95 °C for 2 min, cooled to room temperature, and incubated on ice for 10 min. Electrophoretic Mobility Shift Assay and Determination of the Apparent Dissociation Constants (KD)—The assay mixture (20 l final volume) contained 32P-labeled DNA duplex IV at a concentration of 20 pM and Fpg proteins (0.1–12 nM wild-type Fpg or 1–30 nM FpgK56R, or 1–150 nM FpgK56G, Fpg R53G, or FpgR54G) in buffer B without N-MeIm. The reactions were performed at 0 °C for 10 min and immediately loaded onto prerun non-denaturing 10% PAGE gels as described [21, 22]. If the total concentration of protein [E]0 is close to that of the free protein at equilibrium, KD ϭ [E]0 when 50% of the DNA is bound by the enzyme [21, 22]
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