TY - JOUR
T1 - Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation
AU - Szymanski, Michal R.
AU - Jezewska, Maria J.
AU - Bujalowski, Wlodzimierz
N1 - Funding Information:
We wish to thank Mrs. Gloria Drennan Bellard for reading the manuscript. We would like to thank Dr. Timothy Lohman for comments on this work. This work was supported by National Institutes of Health grants GM-46679 and GM-58565 .
PY - 2011/8/5
Y1 - 2011/8/5
N2 - A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage φX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.
AB - A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage φX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.
KW - DNA priming
KW - DNA replication
KW - fluorescence titrations
KW - motor proteins
KW - protein-DNA interactions
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U2 - 10.1016/j.jmb.2011.05.029
DO - 10.1016/j.jmb.2011.05.029
M3 - Article
C2 - 21641914
AN - SCOPUS:79960700170
SN - 0022-2836
VL - 411
SP - 123
EP - 142
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -