Unwound nature of MAR-capable AT-rich DNA domains (AT-islands) targeted by a highly AT-specific drug bizelesin

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The human genome contains ∼3x10³ of hypervariable minisatellites that consist of up to >3 kbp of 85-100% A/T-rich repeats. These unique domains, termed AT islands, tend to overlap with various sites of genomic instability, including cancer relevant fragile sites FRA16B and FRA16D. AT islands are preferentially targeted by an extremely potent DNA-alkylating antitumor drug bizelesin. Computational analysis suggested that DNA duplexes within AT islands are exceptionally destabilized. This property might underlie the unique potential of AT islands to serve as matrix attachment regions (MARs), the vital elements of nuclear organization. Here we use biochemical approaches and atomic force microscopy (AFM) to characterize the duplexes of cloned model AT islands, including a 1076 bp segment of FRA16B AT island. Partially single-stranded nature of AT-island containing plasmids was demonstrated using the single-strand specific P1 nuclease. P1-induced cleavage (resulting in disappearance of superhelical Form I) was profoundly facilitated in AT-island plasmids relative to control plasmids. For example, P1 at 0.2 Units /reaction converted 48% FRA16B AT island plasmid versus only 6% conversion of the vector plasmid. AFM experiments (conducted in dry mode at pH 7.0) revealed presumably single stranded sections in AT island-containing plasmids that were absent from control non-AT island plasmids. In addition, unusual structural features suggestive of hairpin loops (consistent with palindromic sequences present in the FRA16B domain) were observed in FRA16B plasmids. AFM determinations at elevated pH (to facilitate unwinding) further underscored the profound structural differences between AT island and non-AT island plasmids. Single-strand specific replication protein A (RPA) was used as an alternative way to identify DNA unwinding. AFM demonstrated that RPA formed large aggregates with the FRA16B plasmids but not with non-AT island plasmid. Also, RPA binding to AT island sequences was confirmed using electrophoretic mobility shift assay with linear FRA16B DNA and FRA16B supercoiled plasmid. AFM features indicative of single-stranded sections were markedly reduced in bizelesin-treated AT island plasmids. Likewise, pretreatment with bizelesin abrogated the plasmid’s ability to bind RPA. These observations demonstrate that bizelesin distorts the unusual structures of AT islands. While drug binding is expected to stabilize DNA duplex structure, bizelesin effects on AT island plasmids are probably more complex, as bizelesin-treated plasmids retain enhanced sensitivity to P1 nuclease. Demonstration that AT islands are indeed unwound under physiological conditions corroborates the in silico predictions and warrants further investigations on the role of these domains and their variability in chromatin organization in cancer versus normal cells. (Supported by CA80936)