Escherichia coli DnaA Protein

Abstract

Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA pro-tein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111–148) transiently binds to DnaB as determined by surface plasmon reso-nance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, pro-motes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues. Based on similarities in the repli-cation process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase. The initiation of chromosomal DNA replication in all free-living organisms is a two-stage process that first involves as-sembly of the initiation machinery at specific sequences called the origin of DNA replication (see Ref. 1 for a recent review). Once formed, the initiation complex then assembles proteins that act at the replication fork for semiconservative DNA syn-thesis. In the yeast Saccharomyces cerevisiae, origin sequences are targeted by the six-member origin recognition complex (ORC). 1 ORC in a prereplication complex is then recognized by Cdc6 protein, Cdc45 protein, and minichromosome mainte-nance proteins (reviewed in Ref. 2). The initiation complex is somehow activated by cyclin-dependent kinases and Cdc7-Dbf4 protein kinase, leading to the import of required proteins at the replication forks and DNA synthesis. In Escherichia coli, DnaA protein is the functional counter-part to ORC in that it recognizes specific sequences in the replication origin, oriC. Once bound, it directs formation of the initiation complex through a series of discrete steps. First, it induces a local distortion of an AT-rich region near the left boundary of oriC to form an intermediate, the open complex, in an ATP-dependent process that is assisted by either HU or IHF (3–5). The prepriming complex is then formed by the binding of DnaB helicase from the DnaB-DnaC complex (6, 7). Replication fork assembly and DNA replication follow by the coordinated activities of primase and the ␶ subunit of DNA polymerase III holoenzyme, each forming contacts with DnaB for concerted primer formation, primer extension, and replication fork move-ment (8, 9). Processive DNA synthesis also requires single-stranded DNA-binding protein (SSB) and DNA gyrase to re-lieve positive superhelicity ahead of the replication fork. In the above sequence of events, the entry of DnaB protein at oriC is an important step in the initiation process because this protein is required for bidirectional replication fork movement. How this occurs is poorly understood and limited to the follow-ing observations. First, DnaB protein does not bind efficiently to single-stranded DNA bound by SSB (10). At oriC, DnaA protein induces strand opening in the AT-rich region near the left boundary (3) and also binds to DnaB (11). One model is that DnaA somehow directs the entry of DnaB from the DnaB-DnaC complex to the unwound region covered by SSB so that the helicase is appropriately bound for bidirectional fork move-ment. Recent UV cross-linking studies have shown that DnaC protein can be covalently coupled to ssDNA (12), suggesting the added participation of DnaC at this step. An important issue is to determine the molecular mechanism whereby DnaB protein enters at oriC to establish the replication forks. This may have relevance to the loading of the replicative helicase at replica-tion origins in the eukaryotic cell. The eukaryotic DnaB coun-terpart has not been identified. We recently described a large collection of novel dnaA alleles (13, 14). Their genetic and biochemical characterization indi-cates four functionally distinct domains. One is a nucleotide binding domain carrying a phosphate binding loop (P-loop) within a predicted secondary structure reminiscent of a Ross-mann fold. The second is a region that we speculate is involved in interaction with pSC101-encoded RepA protein. The third is involved in DNA binding. The fourth functional domain is represented by missense mutations clustered near the N ter-minus. These alleles encode proteins that are inactive in DNA replication. The function of this region until now was uncer-tain. We previously thought that the region may participate in oligomerization, because missense mutations that reside in this region were active in DNA binding, but were partially defective in transcriptional repression from the dnaA promoter (14). For DnaA protein to autoregulate its expression, the bind-ing of several protomers to DnaA box sequences in the dnaA regulatory region is required that is speculated to involve oli-gomerization (15). This report examines further the function of the N-terminal domain in initiation of DNA replication. Bio-chemical characterization of deletion mutants lacking this N-terminal region (DnaA⌬62 and DnaA⌬129 lacking residues 1– 62 and 1–129, respectively) revealed that the mutant