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Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP HydrolysisS
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...
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Published in: | The Journal of biological chemistry 2008-12, Vol.283 (52), p.36118-36131 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Online Access: | Get full text |
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Summary: | 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 ∼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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M803158200 |