High resolution characterization of formamidopyrimidine-DNA glycosylase interaction with its substrate by chemical cross-linking and mass spectrometry using substrate analogs.
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.
Rogacheva
Maria
M
Laboratory of Nucleic Acids Chemistry, Department of Chemistry, Moscow State University, Moscow 119992, Russia.
Ishchenko
Alexander
A
Saparbaev
Murat
M
Kuznetsova
Svetlana
S
Ogryzko
Vasily
V
D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008956:Models, Chemical; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010710:Phosphates; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D010710:Phosphates; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D043382:DNA-Formamidopyrimidine Glycosylase