MISSING-BASE AND ETHYLATION INTERFERENCE FOOTPRINTING OF P1 PLASMID REPLICATION INITIATOR

RepA, the replication initiator protein of plasmid P1, binds to specif ic 19 bp sequences on the plasmid DNA. Earlier footprinting studies wi th dimethylsulfate identified the guanines that contact RepA through t he major groove of DNA. In this study, base elimination was used to id entify the contribution of all four bases to the binding reaction. Dep urination and depyrimidation of any base in the neighborhood of the co ntacting guanines was found to decrease RepA binding. These results ar e consistent with the notion that RepA contacts bases of two consecuti ve major grooves on the same face of DNA. We also observed that depuri nation but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA bindi ng upon ethylation. These results indicate that certain bases (e.g. G3 , G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.