The N‐terminus promotes oligomerization of the Escherichia coli initiator protein DnaA

Abstract

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo‐oligomerization of DnaA via its N‐terminus (amino acid residues 1–86) is also essential for initiation. Results from solid‐phase protein‐binding assays indicate that residues 1–86 (or 1–77) of DnaA are necessary and sufficient for self interaction. Using a ‘one‐hybrid‐system’ we found that the DnaA N‐terminus can functionally replace the dimerization domain of coliphage lambda cI repressor: a λcI‐DnaA chimeric protein inhibits λ plasmid replication as efficiently as λcI repressor. DnaA derivatives with deletions in the N‐terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N‐terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N‐termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross‐talk with the nucleotide‐binding domain III or the DNA‐binding domain IV. We propose that E. coli DnaA is composed of largely independent domains — or modules — each contributing a partial, though essential, function to the proper functioning of the ‘holoprotein’.

NMR structure of the N-terminal domain of the replication initiator protein DnaA

“…For insertions, the only exceptions occur in three mycobacterium sequences, in which there is Page 8 12/12/06 an insertion in helix II. Together, these observations indicate that the overall fold of the N-terminal domain is conserved across all species.As was previously noted [5,9,10], the sequence similarity within domain I is lower than within domains III and IV, making it difficult to identify functionally important residues.Experimentally, residues at positions Gln3, Phe4, and Phe7 (Leu5, Trp6 and Cys9 in E.coli) have been shown to be critical for oligomerization [18,19] and residues 24-86, and in particular Pro28, have been implicated in DnaB binding [19,20]. Residues Gln3, Phe4, and Phe7 line the inner face of helix I ( Figure 3) and Pro28 is in the loop connecting helix II and β-strand I.…”

“…Initiation begins only with the ATP-bound form (for reviews see [2][3][4] , as well as domain IV bound to DNA [8] showed that domain III resembles other members of the AAA+ family of ATPases, and that domain IV adopts a helix-turn-helix DNA-binding fold. In contrast, the secondary structure of domain I has been predicted based on multiple sequence alignments [5,9,10], however, little is known about its tertiary structure.DnaA recognizes five high affinity 9 base pair sites as well as several lower affinity 6 base pair sites within OriC via the C-terminal helix-turn-helix DNA-binding domain (domain IV) [11,12]. In addition to direct binding, the formation of oligomers containing 20 to 30 molecules of DnaA at the origin has been demonstrated by electron microscopy [13,14].…”

“…In addition to direct binding, the formation of oligomers containing 20 to 30 molecules of DnaA at the origin has been demonstrated by electron microscopy [13,14]. The mechanism of oligomer formation has been studied extensively [3,15], but the nature of the complex remains poorly understood.Replacement of the dimerization domain of the lambda cI repressor with the N-terminal domain of DnaA resulted in a functional chimeric protein, suggesting direct interactions between the N-terminal domains [10, 16]. Results from solid phase binding assays [10] and gel mobility shift experiments [17] Page 3 12/12/06 have also suggested interactions between N-terminal domains.…”

“…Initiation begins only with the ATP-bound form (for reviews see [2][3][4] , as well as domain IV bound to DNA [8] showed that domain III resembles other members of the AAA+ family of ATPases, and that domain IV adopts a helix-turn-helix DNA-binding fold. In contrast, the secondary structure of domain I has been predicted based on multiple sequence alignments [5,9,10], however, little is known about its tertiary structure.…”

“…Replacement of the dimerization domain of the lambda cI repressor with the N-terminal domain of DnaA resulted in a functional chimeric protein, suggesting direct interactions between the N-terminal domains [10, 16]. Results from solid phase binding assays [10] and gel mobility shift experiments [17] Page 3 12/12/06 have also suggested interactions between N-terminal domains.…”