Chemical footprinting of structural and functional elements of dhfr oriβ during the CHOC 400 cell cycle

Abstract

Oriβ, an origin of replication 3′ to Chinese hamster dihydrofolate reductase ( dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO 4 in vitro and in living cells of three regions within dhfr oriβ which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT 23 dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO 4 modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT 23 repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT 23 repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO 4 in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oriβ adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT 23 repeat and DNA unwinding.