A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry

Abstract

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.

Toward a Detailed Understanding of Base Excision Repair Enzymes: Transition State and Mechanistic Analyses of N-Glycoside Hydrolysis and N-Glycoside Transfer

“…281,354 Fe 4 S 4 is not known to have a direct role in catalysis, but there is some evidence for a role in regulating DNA binding in eMutY. [358][359][360] TthUDGa and PaUDGa steady-state kinetics were complicated by their high affinity for the AP product, with K d ) 9 nM. 281 It was possible to demonstrate activity on U in double and single stranded DNAs, and a preference for mismatches with G. 340 Structures, Mutations, and Catalysis.…”

Domain structure of the DEMETER 5-methylcytosine DNA glycosylase

“…1A) that are properly positioned to interact with a negatively charged DNA backbone (21,25). In addition, two positive arginine residues adjacent to the invariant aspartic acid (Asp-X 4 -Arg-X 3 -Arg) are known to form a hydrogen bond with the FCL loop in Endo III and MutY (25)(26)(27) (Fig. 1A).…”

Mapping of DNA Binding Sites in the Tetrahymena Telomerase Holoenzyme Proteins by UV Cross-Linking and Mass Spectrometry

“…Secondly, we improved an enzymic digestion protocol for cleavage of DNA that is cross-linked to peptides. Unlike a previous procedure, 27,29 our protocol resulted in complete digestion of the DNA, such that only a deoxynucleoside remained attached to a peptide heteroconjugate. The MS/MS spectrum of this peptide could be analyzed with greater accuracy and reliability than spectra of peptides linked to incompletely digested DNA, which were obtained by the previous procedure.…”

“…However, this digestion was found to leave dinucleotides and trinucleotides attached to the peptides, and the MS/MS spectra of these peptides were rather complex and difficult to analyze. 27,29 We added to the digestion protocol a third enzyme, spleen exonuclease, which cleaves deoxynucleoside-3′-monophosphates from DNA chains. As described below, this three-enzyme digestion protocol left only deoxynucleoside attached to the cross-linked amino acids, and as a result, the analysis of the MS/ MS spectra became much simpler.…”