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

Cindy Lou Chepanoske, Olga A. Lukianova, Murielle Lombard, Marie Pierre Golinelli-Cohen, Sheila S. David

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)651-662
Number of pages12
JournalBiochemistry
Volume43
Issue number3
DOIs
StatePublished - Jan 27 2004
Externally publishedYes

Fingerprint

Catalysis
Mass spectrometry
Mass Spectrometry
DNA
Mutagenesis
Repair
DNA Repair
Substrates
Amino Acids
Nucleotide Motifs
Electrospray Ionization Mass Spectrometry
Endonucleases
Deoxyribonuclease I
Cross Reactions
Electrospray ionization
Tandem Mass Spectrometry
Site-Directed Mutagenesis
Sulfur
Alanine
Arginine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chepanoske, C. L., Lukianova, O. A., Lombard, M., Golinelli-Cohen, M. P., & David, S. S. (2004). A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry. Biochemistry, 43(3), 651-662. https://doi.org/10.1021/bi035537e

A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry. / Chepanoske, Cindy Lou; Lukianova, Olga A.; Lombard, Murielle; Golinelli-Cohen, Marie Pierre; David, Sheila S.

In: Biochemistry, Vol. 43, No. 3, 27.01.2004, p. 651-662.

Research output: Contribution to journalArticle

Chepanoske, CL, Lukianova, OA, Lombard, M, Golinelli-Cohen, MP & David, SS 2004, 'A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry', Biochemistry, vol. 43, no. 3, pp. 651-662. https://doi.org/10.1021/bi035537e
Chepanoske CL, Lukianova OA, Lombard M, Golinelli-Cohen MP, David SS. A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry. Biochemistry. 2004 Jan 27;43(3):651-662. https://doi.org/10.1021/bi035537e
Chepanoske, Cindy Lou ; Lukianova, Olga A. ; Lombard, Murielle ; Golinelli-Cohen, Marie Pierre ; David, Sheila S. / A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry. In: Biochemistry. 2004 ; Vol. 43, No. 3. pp. 651-662.
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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.",
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