Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP

Abstract

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5'TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.

Fig. 1.

Location of R boxes and I sites in oriC. (A) Relative position of DnaA-binding sites are marked above the line representing oriC DNA. Additional landmarks, including Factor for inversion stimulation (Fis)- and integration host factor (IHF)-binding sites and the13-mer AT-rich region, are marked below the line. Dark boxes represent I2 and I3. (B) The 9-mer sequences for R boxes and I sites.

Fig. 2.

DnaA-ATP binds preferentially to I sites but not to R boxes. (A) DMS modification patterns after incubating 0 nM DnaA, 100 nM DnaA-ATP, or 100 nM DnaA-ADP with pOC170, measured by primer extension. Sites R5, I1, I2, R4, and I3, and guanines at position 2 or 4 in the recognition site (Fig. 1) are marked. (B) Relative intensities of DMS modification at G2 and G4 within all eight DnaA-binding sites on plasmid incubated with 0 nM DnaA, 30 nM Dna-ATP, 100 nM DnaA-ATP, or 100 nM DnaA-ADP. Sites I1, I3, and R3 lack a guanine in position 2.

Fig. 3.

Single-base changes in positions 1 or 2 in I sites and R boxes reduce DnaA binding. (A) DMS modification patterns after incubating 0 or 100 nM DnaA-ATP with pOC170 or derivative plasmids mutated in position 1 of I2 (pKM10), and I3 (pKM4), or position 2 of I2 (pKM5) and I3 (pKM15). The base changes in the mutants are indicated at the bottom. Sites R5, I1, I2, R4, and I3, and guanines at position 2 and 4 in the recognition site are marked. (B) Relative intensities of DMS modification (G4) for DnaA-binding sites with 0 nM DnaA or 100 nM DnaA-ATP incubated with pOC170 or 100 nM DnaA incubated with mutant plasmids. R1 mutants are pKM16 (T1 to C), and pKM9 (G2 to A). R4 mutants are pKM20 (T1 to G) and pKM17 (G2 to A).

Fig. 4.

Changing G to T at position 3 of the binding site removes discrimination for DnaA-ATP at I2 and I3. (A) DMS modification patterns after incubating 0 nM DnaA, 100 nM DnaA-ATP, or 100 nM DnaA-ADP with plasmids in which G at position 3 of I2 (Left)orI3(Right) is changed to a T. Sites R5, I1, I2, R4, and I3, and guanines at position 2 or 4 in the recognition site (see Fig. 1) are marked. (B) Relative intensities of DMS modified G2 and G4 within all eight DnaA-binding sites.

Fig. 5.

An oriC plasmid mutated in position 3 of I2 and I3 can be unwound with DnaA-ADP, but the region of DNA strand separation is not localized to the 13-mer region. pOC170, or the I2/I3 mutant pKM21 (changing G3 to T) incubated with 0 nM DnaA, 200 nM DnaA-ATP, or 200 nM DnaA-ADP was treated with P1 endonuclease, and cleavage sites were mapped by primer extension. The positions of the right (R), middle (M), and left (L) 13-mer regions, as well as R1 and the promoter for gidA are marked. No cutting induced by DnaA-ATP or DnaA-ADP on either plasmid was detected in oriC to the right of the regions shown.