Regulating DNA replication in bacteria

Abstract

The replication origin and the initiator protein DnaA are the main targets for regulation of chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an active initiation complex can be formed at the origin resulting in strand opening and recruitment of the replicative helicase. In Escherichia coli, oriC activity is directly regulated by DNA methylation and specific oriC-binding proteins. DnaA activity is regulated by proteins that stimulate ATP-DnaA hydrolysis, yielding inactive ADP-DnaA in a replication-coupled negative-feedback manner, and by DnaA-binding DNA elements that control the subcellular localization of DnaA or stimulate the ADP-to-ATP exchange of the DnaA-bound nucleotide. Regulation of dnaA gene expression is also important for initiation. The principle of replication-coupled negative regulation of DnaA found in E. coli is conserved in eukaryotes as well as in bacteria. Regulations by oriC-binding proteins and dnaA gene expression are also conserved in bacteria.

Figure 1.

The replication cycle of the E. coli chromosome in slowly and rapidly growing cells. The chromosomal replication cycle and the cell division cycle in E. coli are shown. When cells are growing slowly (in this example, the doubling time τ is 80 min), cell division occurs after replication of the sister chromosomes is completed. When cells are growing rapidly (i.e., τ is 35 or 25 min), replication initiation simultaneously occurs at each oriC on the partially replicated chromosomes, and cell division occurs after the previous round of replication has been completed. Closed circles, oriC.

Figure 2.

Structures of oriC, datA, and DARSs. (A) The E. coli chromosome is shown as a circle and the locations of oriC (at the genome map position of 84.6 min), dnaA (at 83.6 min), datA (at 94.6 min), DARS1 (at 17.5 min), DARS2 (at 64.0 min), and terC (around 36 min) are indicated. (B) Basic structures of oriC, datA, and DARSs are schematically shown. Closed triangle, DnaA box (9-nucleotide sequence). Gray triangle, I-sites, and τ-sites (both 6-nucleotide sequence). As for datA, DnaA boxes 2 and 3 are most crucial in repression of initiation (Ogawa et al. 2002). For details on low-affinity DnaA binding sites in datA, see Hansen et al. (2007). Open triangle, 13-mer AT-rich motif within DUE. IHF, IHF-binding site; FIS, FIS-binding site; *, GATC sequence within oriC.

Figure 3.

The DnaA cycle and regulation of oriC initiation. Transcription of the dnaA gene is autoregulated. ATP-DnaA is more active in inhibiting dnaA transcription than ADP-DnaA. Also, dnaA transcription increases before replication initiation and is repressed after it in a SeqA-dependent manner. Newly synthesized DnaA molecules adopt the ATP form. ATP-DnaA molecules are also generated by nucleotide exchange of ADP-DnaA in a DARS-dependent manner, though the cell cycle-dependent regulation of DARS remains unclear. With the assistance of DiaA and IHF, ATP-DnaA molecules form a multimeric complex on oriC in a cooperative manner. The resultant oriC complex unwinds DUE and is then engaged in replisome formation. The clamps remain on the synthesized DNA after Okazaki fragments are synthesized and form a complex with ADP-Hda, which interacts with and promotes the hydrolysis of ATP-DnaA (i.e., RIDA). Broken lines indicate expected but unrevealed pathways for cell cycle-coordinated regulation.

Figure 4.

The basic mechanism of RIDA. When DNA polymerase III holoenzyme completes Okazaki fragment synthesis on the lagging strand, the clamp subunit is released from the DNA polymerase III core and remains on the synthesized DNA. ADP-Hda binds to the hydrophobic pocket of the DNA-loaded form of the clamp via the clamp-binding motifs in the amino terminus of Hda. The resultant ADP-Hda-clamp-DNA complex interacts with and promotes the DnaA-bound ATP hydrolysis, releasing ADP-DnaA back into the DnaA cycle. The interaction between the AAA⁺ domains of Hda and DnaA is crucial, and is assisted by the interaction between Hda and DnaA domain IV (DNA-binding domain). N, Hda-amino terminus; I-II, DnaA domain I-II; III, DnaA domain III (AAA⁺ domain); IV, DnaA domain IV (DNA-binding domain).