Cooperativity and Supercoiling Modulate Functions of Human O6-alkylguanine DNA Alkyltransferase

Human cells contain DNA alkyltransferases that protect genomic integrity under normal conditions but also defend tumor cells against chemotherapeutic alkylating agents. Here we explore how structural features of the DNA substrate affect the binding and repair activities of the human O6-alkylguanine-DNA alkyltransferase (AGT). In vitro, cooperative binding results in all-or-nothing association on short templates. A requirement for contact with 4 DNA base-pairs results in oscillation of average binding site size Sapp and cooperativity factor w with template length. Models in which protein molecules overlap along the DNA contour predict that protein-protein contacts will be optimal when the DNA is torsionally relaxed. Supporting this prediction, topoisomerase assays show that AGT binding is accompanied by a small-but-measurable net unwinding (∼7 deg/protein). A torsional free energy model couples this unwinding to observed limits in cooperative cluster size. These results predict that AGT will partition in favor of torsionally-relaxed, relatively protein-free DNA structures like those near replication forks. AGT binds O6-methylG-C and O6-methylG-T lesions with a specificity ratio (KS/KN) far too low for efficient lesion search. This suggests that other factors are needed to direct AGT to lesion sites. Recently we have found that AGT binds the human MutSbeta homologue and PCNA proteins. We propose that these proteins target AGT to sites of mismatch repair (including those containing O6-methylG-T pairs) and to sites near the replication fork, where O6-methylG lesions may be repaired before their potential for mutagenesis can take effect. This work was supported by NIH grant GM070662 to MGF.