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Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins
Author(s) -
Katayama Tsutomu,
Crooke Elliott,
Sekimizu Kazuhisa
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.18050813.x
Subject(s) - dnaa , dnab helicase , biology , seqa protein domain , ter protein , dna replication , replication protein a , replication factor c , helicase , pre replication complex , microbiology and biotechnology , dna , biochemistry , ddb1 , prokaryotic dna replication , dna binding protein , origin of replication , control of chromosome duplication , gene , rna , transcription factor
Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild‐type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild‐type protein. In a system reconstituted with purified proteins at 30°C, the mutant protein initiated replication of single‐stranded DNA that contains a DnaA‐binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA‐binding sequence and functions in the loading of DnaB helicase onto single‐stranded DNA. Thermal stabilities of wild‐type DnaA and DnaAcos activities were comparable. Unlike wild‐type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP‐bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.