A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry.
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.
Chepanoske
Cindy Lou
CL
Department of Chemistry, University of Utah, 315 S 1400 E, Salt Lake City, Utah 84112-0850, USA.
Lukianova
Olga A
OA
Lombard
Murielle
M
Golinelli-Cohen
Marie-Pierre
MP
David
Sheila S
SS
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D020137:Base Pair Mismatch; D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity / Q000235:genetics; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry; D014466:Ultraviolet Rays
C414668:4-thiothymidine; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D001120:Arginine; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000409:Alanine; D013936:Thymidine