Abstract
ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.
Highlights
The unwinding of duplex DNA within the oriC [3, 4]
After synthesis of the Okazaki fragments, the pol III core dissociates, and the sliding clamp remains on the DNA [5]
The third system involves the regulatory inactivation of DnaA (RIDA) and promotes the hydrolysis of DnaA-bound ATP, which yields inactive ADP-DnaA [12, 13]
Summary
Construction and Expression of Hda Derivatives for the N-terminal Analyses—Plasmids pBAD/Hda and pHda (an intermediate pBAD/Hda construction) were constructed previously [25]. pBAD/Hda-cHis was constructed by HindIII digestion and self-ligation of a PCR fragment amplified using pHda as a template and the primers 5Ј-GTCGGATGCGGAAGCTTGGC-3Ј and 5Ј-CCCAAGCTTCAGTGATGGTGATGGTGATGCAACTTCAGAATTTCTTTCACAAACGG-3Ј. Course of analyses, we noticed that the size of Hda expressed by 1C), which is consistent with the results of the Western blot pBAD/Hda (the protein and the plasmid are designated ex-Hda analysis (Fig. 1B) These results support the idea that the CUG sequence uniquely functions as the initiation codon of Hda. The extra N terminus in ex-Hda is rich in hydrophobic residues (Fig. 1A), which might expedite the migration of this protein in SDS-PAGE and might result in a reduced difference in the apparent molecular sizes between ex-Hda and the native Hda. RIDA Activity of Hda-cHis—To analyze the activity of HdacHis in the RIDA system, we overproduced Hda-cHis using pET/Hda-cHis. Approximately 50% of the expressed Hda-cHis was recovered in a soluble cell lysate fraction and was used for purification by nickel-affinity column chromatography. The DNA-loaded clamp was added to a reaction containing ATP-DnaA, Hda, and 2 mM
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