An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin.
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.
Felczak
Magdalena M
MM
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Simmons
Lyle A
LA
Kaguni
Jon M
JM
D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D005976:Glutaral / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004247:DNA; D007930:Leucine; D000409:Alanine; D005976:Glutaral