Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo.
DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
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Mizushima
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Takaki
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Kubota
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Tsuchiya
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Miki
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Katayama
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Sekimizu
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D008239:Lysine