Abstract
Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-Analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-Affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.
| Original language | English |
|---|---|
| Pages (from-to) | 200-211 |
| Number of pages | 12 |
| Journal | Nucleic Acids Research |
| Volume | 48 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 10 2020 |
Funding
Office of the Dean of Research, Georgetown University Medical Center, has provided the funding for the work at GUMC; Department of Energy scientific user facilities supported the small-angle X-ray scattering experiments; Intramural Research Program of CCR, NCI, NIH (in part) supported the in vivo experiments. Funding for open access charge: Office of Dean of Research, Georgetown University Medical Center. Conflict of interest statement. None declared. We would like to thank Dr. Salim Shah, Chief Scientist at Georgetown University, for his help in initiating a collaboration with the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory, Tennessee. We acknowledge laboratory support by the Center for Structural Molecular Biology at Oak Ridge National Laboratory, Tennessee, funded by the Office of Biological and Environmental Research of the U.S. Department of Energy. A portion of this research at ORNL’s Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. We would like to acknowledge Dr. Mark Danielson for the critical reading of manuscript and Julia Grimwade for her advice on P1 endonuclease and DTSSP cross-linking assays, and the anonymous reviewers for thoughtful comments. Author contribution: R.S. conceived the idea and designed the experiments along with E.C. SAXS experiments were performed by R.S., C.B.S. and analyzed by C.B.S. Assistance with designing SAXS experiments were provided by M.J.C. Assistance with protein expression and purification studies were provided by D.P. Experiments in vivo were performed by J.J. and R.S. Molecular modelling studies were performed and analyzed by P.K. Size-exclusion chromatography was performed by R.S., with assistance from K.W. Manuscript was written by R.S., E.C., D.K.C. and C.S. All authors reviewed the results and approved the final version of the manuscript.