An Essential Tryptophan of Escherichia coli DnaA Protein Functions in Oligomerization at the E. coli Replication Origin

Abstract

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.

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

“…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.…”

“…As discussed above, the results from several studies suggested that the N-terminal domain dimerizes or assists in the formation of larger oligomers [10,18,19]. Before initiating NMR studies, we probed the oligomerization state of our construct using both size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC).…”

“…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. In the latter study, the N-terminal domain was shown to be required for cooperative binding of the protein to adjacent DnaA boxes.Mutational analysis has implicated Leu5, Trp6, Glu8, and Cys9 in domain I of E. coli DnaA as being critical for self-oligomerization [18,19]. Other experiments have suggested a role for the ATPase domain in oligomerization [6,7,15, 16,20].…”

“…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.…”

“…Mutational analysis has implicated Leu5, Trp6, Glu8, and Cys9 in domain I of E. coli DnaA as being critical for self-oligomerization [18,19]. Other experiments have suggested a role for the ATPase domain in oligomerization [6,7,15,16,20].…”