Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.
In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding.
Su'etsugu
Masayuki
M
Department of Molecular Biology, Kyushu University Graduate School of Pharmaceutical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Takata
Makoto
M
Kubota
Toshio
T
Matsuda
Yusaku
Y
Katayama
Tsutomu
T
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008954:Models, Biological
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004257:DNA Polymerase II; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli