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Besides the cis-acting oriC site and DnaA, many trans-acting proteins are also required for the initiation of DNA replication, including the DnaB and DnaC proteins. DnaA is required only for initiation, allowing DnaC to load the DnaB helicase for establishing the DNA replication forks. Many proteins used in other cellular functions are also involved, such as the primase (DnaG), the normal RNA polymerase that makes most of the RNA in the cell, and the DNA-binding proteins IHF and Fis (Figure 1.14).

Figure 1.15 outlines how DnaA, DnaB, DnaC, and other proteins participate in the initiation of chromosome replication. As we will see throughout the remainder of this chapter, there are many points at which the initiation of DNA replication is controlled. One important regulatory consideration concerns the nucleotidebinding state of DnaA. While DnaA always binds some of the DnaA boxes, for initiation of DNA replication all of the boxes are bound, forming a special architecture that can open the DNA strands. This type of binding requires that DnaA be bound to ATP (DnaA-ATP). In biology, there are many examples where the nucleotide-bound state of a protein determines its activity. Proteins of this type have the capacity to hydrolyze nucleotides from the NTP to the NDP form, but the energy released is not directly used to actively carry out any particular task and instead allows the configuration of the proteins to change. In the case of DnaA, the ATP-bound form of the protein allows it to form a large multimer structure composed of many molecules of DnaA protein, where the DNA strands are opened through bending of the DNA by DnaA with the help of the IHF and Fis proteins. Within the special complex that productively opens the DNA strands in the origin, DnaA binding appears to take on a different form when it interacts with DUE (DNA unwinding element), preferentially engaging single-strand DNA in this region to facilitate strand opening (Figure 1.15). The binding and opening are also aided by supercoiling at the origin (see “Supercoiling” below) and by the SSB protein, which helps to keep the helix from reforming. DnaA binds directly to the helicase DnaB, and in a process involving DnaC, DnaB helicase is loaded onto oriC. Action of the DnaB helicase opens the strands further for priming and replication, and DnaC leaves the complex.