A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.
Nucleic Acids Res
; 48(1): 200-211, 2020 01 10.
Article
en En
| MEDLINE
| ID: mdl-31665475
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
ADN Bacteriano
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Regulación Bacteriana de la Expresión Génica
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Adenosina Difosfato
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Adenosina Trifosfato
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Proteínas de Unión al ADN
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Replicación del ADN
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Escherichia coli
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos