Structural insights into the repair mechanism of AGT for methyl-induced DNA damage

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

Activation by NarL at the Escherichia coli ogt promoter

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.