High Resolution Characterization of Formamidopyrimidine-DNA Glycosylase Interaction with Its Substrate by Chemical Cross-linking and Mass Spectrometry Using Substrate Analogs

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.Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) 2 (also known as MutM) and human 8-oxoguanine-DNA glycosylase (hOgg1) are DNA repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (8-oxoG) residues from DNA (1-3). 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 G⅐C 3 T⅐A transversions in vivo and in vitro (5, 6). Thus the Fpg and hOgg1 DNA glycosylases are important in prevention of the mutagenic effects of 8-oxoG residues present in DNA and maintenance of genome integrity. To date, most studies have focused on the interactions of Fpg and hOgg1 with oxidized bases, and little attention has been paid to contacts with phosphate groups in damaged DNA. However, it is evident that interactions of the DNA glycosylases with DNA phosphate groups are critical for the target search along DNA and base lesion recognition. It has been shown that the interaction of Fpg and hOgg1 with damaged DNA causes significant conformational changes in helix structure, such as partial denaturation, widening of the minor groove, sharp kinking of the DNA backbone at the lesion site, and flippi...

Thiopurine Derivative-Induced Fpg/Nei DNA Glycosylase Inhibition: Structural, Dynamic and Functional Insights

“…This DNA bending facilitates the extrusion of the damaged nucleoside outside the DNA helix and its stabilization inside the active site pocket that exposes it for catalysis [28,36,40]. In addition to its role in DNA binding, K57 is also proposed as an accessory catalytic residue for DNA glycosylase activity of the EcFpg [38,41,42]. Through binding at site II, the present crystal structures indicate that 2TX/TXn in their reduced forms do not disturb the local structure of the complex (the DNA backbone conformation and the side chain orientation of R260 and K57 are unchanged).…”

Using Shifts in Amino Acid Frequency and Substitution Rate to Identify Latent Structural Characters in Base-Excision Repair Enzymes

“…Identification of highly conserved amino acids usually serves to find these first-shell amino acids [70], [75], [76], [77], [78], [79], [80]. Collections of second- and third-shell amino acids stabilize first-shell amino acids.…”

Structure and function analysis of protein–nucleic acid complexes

“…Rogacheva et al 113 used a combination of cross-linking and high-resolution mass spectrometry to analyze contacts between DNA phosphate groups and the active site residues of the excision repair enzymes, E. coli formamidopyrimidine DNA glycosylase (Fpg) and its eukaryotic analogue Ð human 8-oxoguanine DNA glycosylase (hOGG1). These enzymes are bifunctional N-glycosylases/AP-lyases that catalyze excision of 8-oxoguanine residues and subsequent cleavage of the sugar ± phosphate backbone of DNA.…”

“…On the basis of the analysis of the experimental data, models were constructed for the interaction of Fpg and hOGG1 with phosphate groups of damaged DNA within the specific enzyme ± substrate complex in solution. 113 The authors suggested that this approach can be employed to study prokaryotic and eukaryotic homologues of 8-oxoguanine DNA glycosylases, as well as other repair enzymes.…”

“…Formation of a covalent bond between an amino acid of a protein and a phosphate group of DNA within a specific enzyme ± substrate complex. 113 The development of the combined UV cross-linking ± immunoprecipitation method can also be employed in structural studies of protein ± nucleic acid complexes in vivo. 116 The authors mentioned that UV light as a zerolength cross-linking reagent causes less distortion in the structure of the complex under study compared to chemical reagents.…”