Fig. 6.

DUE activity of oriC with deletions between R1 and the IHF-binding site. (A) DNA sequence covering from R1 to R5M. WT sequence and deleted sequences (d5–d10) are shown. Sequences of the R1, R5M, and IHF-binding site (IBS) are highlighted by red, cyan, and green, respectively. Deleted sequences are indicated by hyphens. DUE activities of each oriC are also shown based on the data shown in B and C. +, WT level; −, inactive. (B and C) DUE assay. The given amounts of ATP/ADP–DnaA were incubated with plasmid M13oriCMS9 or its derivatives bearing deletions as shown in A, followed by analysis using ssDNA-specific nuclease P1, EcoRI, and 1% agarose gel electrophoresis (B). Percentages of P1 nuclease-digested oriC DNA molecules per that of input DNA are shown as “DUE unwinding (%)” (C). (D) Positional examination of the R1-bound DnaA in the R1–IBS region-deleted oriC. In a representative structure of left-half subcomplex, the R1-bound DnaA domain III–IV was relocated toward the IHF-binding site by 5 base pair (bp) (orange) or 8 bp (red).