Site-Directed Mutagenesis of the Cysteine Ligands to the [4Fe−4S] Cluster of Escherichia coli MutY

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.

DNA-mediated charge transport for DNA repair

“…To probe this question, we examined a mutant protein, C199H, also expressed as a fusion with MBP, in which Cys-199 is mutated to a histidine. C199H has similar activity, substrate specificity, and structure as the WT, except for histidine ligation of the cluster at position 199 (17,65,66). EPR and crystallography indicate that coordination to the histidine is somewhat destabilized, leading to loss of Fe with the partial formation of [3Fe4S] clusters.…”

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

“…25,32,33 In these experiments, the absence of MutY activity allows spontaneous mutations in the rifampicin binding site of E. coli RNA polymerase to accumulate and render rifampicin less effective as a block to transcription. 14,34,35 However, such assays are not readily adaptable to evaluating the repair of a specific damaged DNA substrate or unnatural synthetic DNA nucleotides.…”

An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases

“…A variety of biochemical and biophysical studies suggests that the 4Fe4S cluster in these enzymes is not involved directly in catalysis (24). Instead it functions as a structural cross-link analogous to disulfide bonds or zinc fingers and nonetheless contributes to substrate recognition by maintaining the structure of protein segments involved in DNA interactions (25)(26)(27). On the basis of the structural homology between the Family 4 enzymes and the Family 2 bacterial MUG, the deduced site of the 4Fe4S cluster in Pa-UDG suggests that it would not participate directly in glycosylase activity.…”