Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY

Marie Pierre Golinelli, Nikolas H. Chmiel, Sheila S. David

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine:2'- deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7022-7025]. MutY prevents DNA mutations resulting from the misincorporation of A opposite OG by using N-glycosylase activity to remove the adenine base. An interesting feature of MutY is that it contains a [4Fe-4S]2+ cluster that has been shown to play an important role in substrate recognition [Porello, S. L., Cannon, M. J., David, S.S. (1998) Biochemistry 37, 6465-6475]. Herein, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, and alanine. The extent to which the various mutations reduce the levels of protein overexpression suggests that coordination of the [4Fe-4S]2+ cluster provides stability to MutY in vivo. The ability of the mutated enzymes to bind to a substrate analogue DNA duplex and their in vivo activity were evaluated. Remarkably, the effects are both substitution and position dependent. For example, replacement of cysteine 199 with histidine provides a mutated enzyme that is expressed at high levels and exhibits DNA binding and in vivo activity similar to the WT enzyme. These results suggest that histidine coordination to the iron-sulfur cluster may be accommodated at this position in MutY. In contrast, replacement of cysteine 192 with histidine results in less efficient DNA binding and in vivo activity compared to the WT enzyme without affecting levels of overexpression. The results from the site-directed mutagenesis suggest that the structural properties of the iron-sulfur cluster coordination domain are important for both substrate DNA recognition and the in vivo activity of MutY.

Original languageEnglish (US)
Pages (from-to)6997-7007
Number of pages11
JournalBiochemistry
Volume38
Issue number22
DOIs
StatePublished - Jun 1 1999
Externally publishedYes

Fingerprint

Mutagenesis
Site-Directed Mutagenesis
Escherichia coli
Cysteine
Ligands
Histidine
DNA
Enzymes
Sulfur
Substrates
Iron
DNA Repair Enzymes
Mutation
Biochemistry
Adenine
Alanine
Serine
Structural properties
Repair
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY. / Golinelli, Marie Pierre; Chmiel, Nikolas H.; David, Sheila S.

In: Biochemistry, Vol. 38, No. 22, 01.06.1999, p. 6997-7007.

Research output: Contribution to journalArticle

Golinelli, Marie Pierre ; Chmiel, Nikolas H. ; David, Sheila S. / Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY. In: Biochemistry. 1999 ; Vol. 38, No. 22. pp. 6997-7007.
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