DiaA dynamics are coupled with changes in initial origin complexes leading to helicase loading.
Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.
Keyamura
Kenji
K
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Abe
Yoshito
Y
Higashi
Masahiro
M
Ueda
Tadashi
T
Katayama
Tsutomu
T
D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002875:Chromosomes / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D053488:DnaB Helicases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D051738:Origin Recognition Complex; D010957:Plasmids / Q000378:metabolism; D013696:Temperature
D002352:Carrier Proteins; C523634:DiaA protein, E coli; D051738:Origin Recognition Complex; D000255:Adenosine Triphosphate; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I; D004265:DNA Helicases; D053488:DnaB Helicases