DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

Abstract

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O 6 -alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O 6 -methylguanine (O 6 meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

Structural studies of MJ1529, an O6-methylguanine-DNA methyltransferase

“…Interaction of this region of the protein with DNA is consistent with chemical shift mapping data on AdaC, in which significant shifts of amide peaks upon titration with DNA occurred in helix e and helix f, and the wing residues (figure 7.b) 19,33 .…”

“…Protein/DNA interfaces are proposed to be somewhat flexible 19,44,45 . This would seem to be particularly important for DNA-repair proteins such as MGMT, that have little sequence specificity, little preference for double over single-stranded DNA, and can accommodate different sizes and types of base modifications.…”

“…Support for this mode of repair by MGMT is provided by chemical shift mapping studies of AdaC, indicating that the structure is not greatly perturbed upon binding 19 . However, direct physical evidence for base-flipping by MGMT has been difficult to obtain due to the fast nature of the reaction.…”

“…Binding constant studies put dissociation constants (K d ) for MGMT-DNA complexes in the low (0.5-20) micromolar range for both methylated and unmethylated DNA 10,11,[19][20][21] . This moderate affinity for DNA and low preference for methylated over unmethylated DNA in particular may be reflected in the low sequence specificity of repair.…”

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

“…We cloned the expression plasmids of the C-terminal domain of Ada (C-Ada) and AlkA by incorporating the two genes into pET28a vector, and then the expression and purification of the two recombinant proteins followed known procedures (37,38). The DNA oligonucleotides used in the enzymatic assays were 5Ј-TAGACATTGCCATTCTCGATA-GG(m 1 A)TCCGGTCAAACCTAGACGAATTCCA-3Ј or 5Ј-ATTGCCATTCTCGATAGG(m 1 A)TCCGGTCAAACCTA-GACGAA-3Ј for AlkB or ALKBH3 repair, 5Ј-TGGAATTCG-TCTAGGTTTGACCGGATCCTATCGAGAATGGCAATG-TCTA-3Ј or 5Ј-TTCGTCTAGGTTTGACCGGATCCTATC-GAGAATGGCAAT-3Ј as the complementary DNA sequence for the duplex substrates of AlkB or ALKBH2 repair, 5Ј-GCC-ATTCTCGATAGGCGCA(O 6 mG)CTGAGCTCGCGTCCG-GTCA-3Ј complementary to 5Ј-TGACCGGACGCGAGCTC-AGCTGCGCCTATCGAGAATGGC-3Ј for Ada repair, and 5Ј-CGATAGCATCCTGCCTTCTCTCCAT-3Ј complementary to 5Ј-ATGGAGAGAAGGAAGGATGCTATCG-3Ј for AlkA repair, respectively.…”