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

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.

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

“…Many DNA repair proteins have a tendency to bind to destabilized DNA fragment ends, as discussed previously (62). For UvrB(C) and XPD(/p44), end binding preference varied strongly between experiments from 0 to 54% (with averages of 9% for UvrB and XPD alone, 4% for UvrBC, and 22% for XPD/p44).…”

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

“…Enhanced binding of a protein to a specific target site within a DNA fragment results in a peak in the distribution at the corresponding position: 0% for complexes bound at DNA fragment ends, 50% for complexes bound at a DNA fragment center, ϳ31% for complexes bound at the lesion, and/or DNA bubble for specific DNA substrates in these experiments. Many proteins involved in DNA repair show a propensity to also bind to DNA fragment ends, likely as a result of local helix destabilization (29,32). XPD also shows a slight preference for DNA ends (bars at 0% DNA length in position distributions of Fig.…”

Insight into the cooperative DNA binding of the O6-alkylguanine DNA alkyltransferase

“…For AGT binding to long DNA substrates of 1,000 and ~2,700 bp length, values of s = (6.81 ± 0.14) and (6.32 ± 0.12) bp, K = (7960 ± 916) and (9667 ± 1499), and ω = (44.2 ± 3.8) and (35.9 ± 6.8) were obtained, respectively, from sedimentation equilibrium analytical ultracentrifugation data [7]. The observed limiting binding site sizes were larger than the value of ~4 bp/protein obtained with shorter DNA oligonucleotides [26].…”

“…This likely reflects packing inhomogeneities that occur when the substrate is large and which are largely absent near binding saturation, when short substrates are used. The dependence of cluster size C̄ on AGT concentration predicted by McGhee-von Hippel theory using these values of s , K , and ω is shown in Figure 3 [7]. …”

“…AFM imaging allowed the direct characterization of AGT complexes with long DNA substrates (1,000 and ~2,700 bp length, same as above; Figure 4) [7]. Since these DNA substrates did not contain any introduced O 6 -methylguanine sites, binding should be dominated by non-specific interactions.…”