Figure 1

Key systems maintaining the active and unmethylated state of DNA at sequences with the innate potential for non-B DNA structure formation. In this enzymologically-based model, non-B DNA structure forms spontaneously or in response to replication stress or carcinogen-linked damage, inducing DNA methylation de novo[3, 4, 103, 104]. The three-nucleotide recognition motif [4] of DNMT1 (C:G-C) is highlighted in the schematic of the non-B structure in the upper right of the figure. Helicase resolution at non-B structures produces hemimethylated DNA. Hypermethylation is prevented by the action of TET dioxygenase on its preferred hemimethylated substrate [17]. When stress overwhelms the capacity of TET dioxygenase to hydroxymethylate hemimethylated DNA in the affected region, hypermethylation will result. In this model, helicase lesions, DNMT lesions or TET dioxygenase lesions are expected to generate chromosome instability and the selective induction of fragile sites. Methylated unusual DNA structures that are not resolved by helicase action may be removed by excision repair-linked pathways where the unmethylated state is restored by DNA synthesis. (A) Molecular model of the hypermethylated C-rich strand hairpin formed at fragile site FRAXA. The model was constructed in Biograf 3.1 (Molecular Simulations Inc, San Diego, CA, USA) and rendered with the UCSF Chimera package (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, CA, USA). It is based on NMR data presented by Chen et al.[20]. (B) Activity of human DNA methyltransferase 1 (hDNMT1) on hemimethylated DNA and hemihydroxymethylated DNA. hDNMT1 was purified from nuclear extracts [105] prepared from cultured HeLa S3 cells. Purification and enzyme activity measurements were carried out as previously described [106, 107]. The purified enzyme had a specific activity of 20.96 fmole³HCH₃/min/mg. Duplex ODN substrates were synthesized and annealed as previously described [107]. The results confirm the findings of Hashimoto et al. [17] with cloned DNMT1.