Cooperative Cluster Formation, DNA Bending and Base-Flipping by O⁶-Alkylguanine-DNA Alkyltransferase

O-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O-alkylguanine and O-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 DNAreplication complexes. The low sequence specificity allows efficient and unbiased lesion search across