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Interaction of the Sliding Clamp ß-Subunit and Hda, a DnaA-Related Protein

Mareike Kurz, Brian Dalrymple, Gene Wijffels, and Kritaya Kongsuwan^*

CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia Queensland Dominion 4067, Australia

Received 23 October 2003/ Accepted 12 February 2004

In Escherichia coli, interactions between the replication initiation protein DnaA, the ß subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and ß proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with ß in vitro. A new ß-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified ß-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind ß. A 10-amino-acid peptide containing the E. coli Hda ß-binding motif was shown to compete with Hda for binding to ß in an Hda-ß interaction assay. These results establish that the interaction of Hda with ß is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Present address: Institute of Molecular Biosciences, University of Queensland, St. Lucia QLD 4067, Australia.

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