Positively charged residues within the iron-sulfur cluster loop of E. coli MutY participate in damage recognition and removal

Cindy Lou Chepanoske, Marie Pierre Golinelli, Scott D. Williams, Sheila S. David

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Escherichia coli MutY is an adenine glycosylase involved in base excision repair that recognizes OG:A (where OG = 7,8-dihydro-8-oxo-2'-deoxyguanosine) and G:A mismatches in DNA. MutY contains a solvent-exposed polypeptide loop between two of the cysteine ligands to the [4Fe-4S]2+ cluster, referred to as the iron-sulfur cluster loop (FCL) motif. The FCL is located adjacent to the proposed active site pocket and has been suggested to be part of the DNA binding surface of MutY (Y. Guan et al., 1998, Nat. Struct. Biol. 5, 1058-1064). In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysine 196, and lysine 198 with alanine on the enzymatic properties of MutY. The properties of the R194A, K196A, and K198A enzymes were also compared to the properties of mutated enzymes in which lysine residues near the active site pocket were replaced with alanine or glycine. Substrate recognition was evaluated using a duplex containing a 2'-deoxyadenosine analog in a base pair opposite G or OG. These results indicate that removal of positively charged amino acids within the FCL and the active site compromise the ability of the enzyme to bind to the substrate analog. However, only the K198A enzyme exhibited a significant reduction (15-fold) of the rate of adenine removal from a G:A base pair-containing duplex. This is the first direct evidence that Lys 198 within the FCL motif of MutY has a role in specific damage recognition and removal. Furthermore, these results suggest that the FCL motif is intimately involved in the base removal process. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)11-19
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume380
Issue number1
DOIs
StatePublished - Aug 1 2000
Externally publishedYes

Fingerprint

Sulfur
Escherichia coli
Iron
Lysine
Catalytic Domain
Enzymes
Base Pairing
Alanine
DNA
Adenine
Substrates
DNA Repair
Glycine
Cysteine
Arginine
Repair
Ligands
Amino Acids
Peptides

Keywords

  • 8-oxoguanine
  • Base excision repair
  • DNA damage
  • DNA recognition
  • DNA repair
  • Glycosylase
  • Iron-sulfur protein

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Positively charged residues within the iron-sulfur cluster loop of E. coli MutY participate in damage recognition and removal. / Chepanoske, Cindy Lou; Golinelli, Marie Pierre; Williams, Scott D.; David, Sheila S.

In: Archives of Biochemistry and Biophysics, Vol. 380, No. 1, 01.08.2000, p. 11-19.

Research output: Contribution to journalArticle

Chepanoske, Cindy Lou ; Golinelli, Marie Pierre ; Williams, Scott D. ; David, Sheila S. / Positively charged residues within the iron-sulfur cluster loop of E. coli MutY participate in damage recognition and removal. In: Archives of Biochemistry and Biophysics. 2000 ; Vol. 380, No. 1. pp. 11-19.
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abstract = "Escherichia coli MutY is an adenine glycosylase involved in base excision repair that recognizes OG:A (where OG = 7,8-dihydro-8-oxo-2'-deoxyguanosine) and G:A mismatches in DNA. MutY contains a solvent-exposed polypeptide loop between two of the cysteine ligands to the [4Fe-4S]2+ cluster, referred to as the iron-sulfur cluster loop (FCL) motif. The FCL is located adjacent to the proposed active site pocket and has been suggested to be part of the DNA binding surface of MutY (Y. Guan et al., 1998, Nat. Struct. Biol. 5, 1058-1064). In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysine 196, and lysine 198 with alanine on the enzymatic properties of MutY. The properties of the R194A, K196A, and K198A enzymes were also compared to the properties of mutated enzymes in which lysine residues near the active site pocket were replaced with alanine or glycine. Substrate recognition was evaluated using a duplex containing a 2'-deoxyadenosine analog in a base pair opposite G or OG. These results indicate that removal of positively charged amino acids within the FCL and the active site compromise the ability of the enzyme to bind to the substrate analog. However, only the K198A enzyme exhibited a significant reduction (15-fold) of the rate of adenine removal from a G:A base pair-containing duplex. This is the first direct evidence that Lys 198 within the FCL motif of MutY has a role in specific damage recognition and removal. Furthermore, these results suggest that the FCL motif is intimately involved in the base removal process. (C) 2000 Academic Press.",
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AB - Escherichia coli MutY is an adenine glycosylase involved in base excision repair that recognizes OG:A (where OG = 7,8-dihydro-8-oxo-2'-deoxyguanosine) and G:A mismatches in DNA. MutY contains a solvent-exposed polypeptide loop between two of the cysteine ligands to the [4Fe-4S]2+ cluster, referred to as the iron-sulfur cluster loop (FCL) motif. The FCL is located adjacent to the proposed active site pocket and has been suggested to be part of the DNA binding surface of MutY (Y. Guan et al., 1998, Nat. Struct. Biol. 5, 1058-1064). In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysine 196, and lysine 198 with alanine on the enzymatic properties of MutY. The properties of the R194A, K196A, and K198A enzymes were also compared to the properties of mutated enzymes in which lysine residues near the active site pocket were replaced with alanine or glycine. Substrate recognition was evaluated using a duplex containing a 2'-deoxyadenosine analog in a base pair opposite G or OG. These results indicate that removal of positively charged amino acids within the FCL and the active site compromise the ability of the enzyme to bind to the substrate analog. However, only the K198A enzyme exhibited a significant reduction (15-fold) of the rate of adenine removal from a G:A base pair-containing duplex. This is the first direct evidence that Lys 198 within the FCL motif of MutY has a role in specific damage recognition and removal. Furthermore, these results suggest that the FCL motif is intimately involved in the base removal process. (C) 2000 Academic Press.

KW - 8-oxoguanine

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KW - DNA recognition

KW - DNA repair

KW - Glycosylase

KW - Iron-sulfur protein

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