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Mutations that probe the cooperative assembly of O⁶-alkylguanine-DNA alkyltransferase complexes
Claire A Adams1, Michael G Fried
1Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, United States.
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.
Insights
O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA damage from alkylating agents. Mutations disrupting protein-protein interactions reduced AGT
Area of Science:
- Biochemistry
- Molecular Biology
- DNA Repair Mechanisms
Background:
- O(6)-Alkylguanine-DNA alkyltransferase (AGT) is crucial for repairing DNA damage caused by alkylating agents.
- AGT functions by repairing mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts.
- Models suggest AGT binds DNA cooperatively, involving specific residues from adjacent protein molecules.
Purpose of the Study:
- To investigate the role of specific residues in AGT's cooperative DNA binding and protein-protein interactions.
- To test predictions from models of cooperative AGT-DNA complexes.
Main Methods:
- Directed mutagenesis was employed to substitute native residues with alanine triplets in predicted interface regions (residues 1-7 and 163-169).
- Mutant AGT proteins were expressed and analyzed for proper folding, DNA binding stoichiometry, and protective capacity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
- DNA binding cooperativity and affinity were assessed for wild-type and mutant AGTs.
Main Results:
- Six of eight designed AGT mutants were successfully expressed and exhibited proper folding and DNA binding stoichiometry.
- All expressed mutants showed reduced DNA binding cooperativity and affinity compared to wild-type AGT.
- Specific mutants (KDC(3-5)-AAA, DCE(4-6)-AAA, KEW(165-167)-AAA) conferred increased susceptibility to MNNG, indicating impaired DNA repair.
- This study provides the first evidence linking protein-protein interfaces and cooperative interactions to AGT's protective function.
Conclusions:
- Protein-protein interactions at the AGT interface are intrinsically coupled with protein-DNA interactions.
- Disrupting these interfaces significantly impairs AGT's DNA repair efficiency and cellular protection.
- Cooperative protein-protein interactions play a vital role in the functional mechanism of AGT in DNA repair.
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