Cooperative cluster formation, DNA bending and base-flipping by O 6-alkylguanine-DNA alkyltransferase

Ingrid Tessmer, Manana Melikishvili, Michael G. Fried

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of 11 proteins. Longer clusters, predicted by the McGhee-von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (∼0, ∼30 and ∼60°) that are consistent with models in which each protein induces a bend of ∼30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.

Original languageEnglish
Pages (from-to)8296-8308
Number of pages13
JournalNucleic Acids Research
Volume40
Issue number17
DOIs
StatePublished - Sep 2012

Bibliographical note

Funding Information:
Deutsche Forschungsgemeinschaft [Forschungszentrum FZ82 to I.T.]; National Institutes of Health (NIH) [GM-070662 to M.G.F.]. Funding for open access charge: NIH.

ASJC Scopus subject areas

  • Genetics

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