title: Mutations that probe the cooperative assembly of O⁶-alkylguanine-DNA alkyltransferase complexes.
abstract: O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule and residues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able to protect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced the affinity of AGT for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residues at the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-protective mechanisms of AGT.
authors.affiliation: Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, United States.
authors.forename: Claire A
authors.initials: CA
authors.lastname: Adams
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase