Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

Abstract

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6‐methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self‐methylation of the active site cysteine renders the protein inactive. Here we report the X‐ray structure of the 19 kDa C‐terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases

“…This confirms previous studies in which crude cell extracts of E. coli were used (10 -13) and rules out the possibility that the resistance is due to the rapid metabolism of the O 6 -benzylguanine by bacterial enzymes. The crystal structure of Ada-C suggests that the tryptophan residue at position 336 may limit access to the cysteine acceptor site (6,7). Studies with the human AGT have indicated that a proline residue located 5 residues to the amino side of this cysteine is necessary for optimal reaction with O 6 -benzylguanine (22,23).…”

“…6 -Benzylguanine-The purified alkyltransferase proteins were incubated in 50 mM Tris-HCl, pH 7.5, 0. 5 mM dithiothreitol, and 0.1 mM EDTA with 5Ј-d(AACAGCCATATa 6 GGCCC)-3Ј in which a 6 G represents O 6 -benzylguanine or O 6 -methylguanine.…”

“…The E. coli Ada protein is the best characterized of all of the alkyltransferases, and a crystal structure at 2.1-Å resolution for the carboxyl-terminal domain of this protein (Ada-C), 1 which is fully active in repair of O 6 -methylguanine, has been published (6,7). This structure shows that the cysteine acceptor site is buried, and the authors suggest that the protein must undergo a significant conformational change upon binding a DNA substrate and that this alteration facilitates the transfer reaction.…”

Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine‒DNA alkyltransferase insensitive to O6-benzylguanine

“…These highly conserved amino acids are located in the carboxyl half of the protein, which includes the DNA-binding domain and the cysteine acceptor site [9][10][11] (Figure 1). Based on the alkyltransferase crystal structure [12][13][14], models of the binding of the DNA substrate to the protein, and studies of the reaction mechanism [9,15,16], it is likely that the O'-alkylguanine residue is flipped out of the DNA helix and bound in an active-site pocket containing the Cys-145 acceptor.…”

DNA Binding Specificity of Mu Transcription Factor C and Crystallization of C : DNA Complex

“…In eukaryotes the HTH motif is seen in proteins that regulate development and differentiation, transcription factors (TFIIB/ TFIIE) and chromatin proteins (histone H1) (Schultz et al, 1991;Wilson et al, 1992Brennan, 1993Clark et al, 1993;Ramakrishnan et al, 1993;Swindells, 1995;Kodandapani et al, 1996;Aravind and Koonin, 1999;Gajiwala and Burley, 2000). This motif has also been identified in proteins involved in DNA repair and RNA metabolism (Moore et al, 1994;Wah et al, 1997;Selmer and Su, 2002;Alfano et al, 2004;Dong et al, 2004). In addition to DNA binding, the HTH can also be adapted for mediating specific protein-protein interactions or can be part of a structural unit of a large enzymatic domain (Guo et al, 1997;Wong et al, 2000;Zheng et al, 2002).…”