DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA

Kenji Keyamura,Tsutomu Katayama

doi:10.1074/jbc.m111.233403

Abstract

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

Highlights

Ministry of Education, Culture, Sports, Technology and Science of Japan. □S The on-line version of this article contains supplemental Fig. 1. 1 To whom correspondence should be addressed: Dept. of Molecular Biology, within oriC, facilitates formation of an open complex [3]
DnaA Leu-422 and Pro-423 residues within DnaA domain IV were required for regulatory inactivation of DnaA (RIDA) activity and interaction with Hda in vitro, which is consistent with the results of in vivo analyses. These amino acid residues did not play a crucial role in the process of DNA replication initiation. These findings suggest that cross-talk between DnaA domain IV and the Hda AAAϩ domain is required for the formation of an active RIDA complex
The resulting model showed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV would reside on an interface with the Hda AAAϩ domain (Fig. 1, B–D)

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—The E. coli K12 derivatives KH5402-1 (thyA) and WM433 (dnaA204) have been described previously [17, 22]. DnaA-Hda Interaction Using SPR Analysis—The Hda binding activity of DnaA proteins was determined by SPR analysis using an NTA sensor chip (Biacore) Buffers used for this analysis were as follows: (i) regeneration buffer (10 mM Hepes-KOH (pH 8.3), 150 mM NaCl, 350 mM EDTA, and 0.005% Surfactant P20; (ii) wash buffer (10 mM Tris-HCl (pH 7.6), 300 mM NaCl, 1 mM EDTA, and 0.05% SDS; (iii) nickel buffer (10 mM HepesKOH (pH 7.6), 150 mM NaCl, 50 ␮M EDTA, 0.01% Brij-58, and 5 mM NiCl2; and (iv) eluent buffer (10 mM Hepes-KOH (pH 7.6), 150 mM potassium acetate, 5 mM magnesium acetate, 50 ␮M EDTA, 0.01% Brij-58, 8 mM 2-mercaptoethanol, and 0.1 mM ADP). Radiolabeled nucleotides were separated using thin-layer chromatography and quantified using an imaging analyzer

RESULTS

Am ϩ ϩ ϩ ϩ

DISCUSSION