Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jan;13(1):301-5.
doi: 10.1110/ps.03319404.

Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

Joseph J Rasimas  1 , Paula A Dalessio, Ira J Ropson, Anthony E Pegg, Michael G Fried
Affiliations

Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

Joseph J Rasimas et al. Protein Sci. 2004 Jan.

Abstract

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
The unfolding of nonalkylated AGT (open circles) and alkylated AGT (closed squares) at equilibrium detected by circular dichroism. The final protein concentration was 110 μg/mL. Data were normalized to a full-scale of 1.0. (Inset) A plot of ΔG° vs. urea concentration for 1.5 M ≤ [urea] ≤ 6 M.
Figure 2.
Figure 2.
Effect of urea on the stability of an AGT–DNA complex. (Top panel) The complex of the nonalkylated AGT with the 16-mer duplex, titrated with urea. Samples contained 0.6 × 10−7 M 16-mer duplex and 1.9 × 10−6M nonalkylated AGT, in binding buffer modified with 0–7.6 M urea (lanes a–k). (Bottom panel) The complex of benzylated AGT with the 16-mer duplex titrated with urea. Samples contained 0.6 × 10−7 M 16-mer duplex and 2.1 × 10−6M benzylated AGT, in binding buffer modified with 0–6.8 M urea (lanes a–k).
Figure 3.
Figure 3.
Analysis of the effect of urea on the stability of an AGT–DNA complex. Graph of mole fraction of 16-mer DNA bound as a function of [urea]. (Open symbols) Complex formed with nonalkylated AGT; (solid symbols) complex formed with benzylated AGT. The different symbols within a data set (e.g., open circles, squares) correspond to independent experiments. (Inset) Graph of ΔG° as a function of urea concentration.
See this image and copyright information in PMC

References

    1. Dalessio, P.M. and Ropson, I.J. 1998. pH dependence of the folding of intestinal fatty acid binding protein. Arch. Biochem. Biophys. 359 199–208. - PubMed
    1. Daniels, D.S. and Tainer, J.A. 2000. Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O6-alkylguanine-DNA alkyltransferase. Mutation Res. 460 151–163. - PubMed
    1. Daniels, D.S., Mol, C.D., Arval, A.S., Kanugula, S., Pegg, A.E., and Tainer, J.A. 2000. Active and alkylated human AGT structures: A novel zinc site, inhibitor and extrahelical binding. DNA damage reversal revealed by mutants and structures of active and alkylated human AGT. EMBO J. 19 1719–1730. - PMC - PubMed
    1. Fried, M.G. 1989. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10 366–376. - PubMed
    1. Fried, M.G. and Crothers, D.M. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9 6505–6525. - PMC - PubMed

Publication types

LinkOut - more resources