Fig. 4.
The role of the 5′ triphosphate in primer length determination. (A) Nucleotide competition binding by PriX, measured by fluorescence polarization. Fluorescein-12-ATP and PriX are at fixed concentrations of 20 nM and 2.5 µM, respectively. Concentrations of unlabeled ATP, AMP, or adenosine range from 0 to 2 mM. (B) Primase substrate binding measured by fluorescence polarization. 5′ FAM-labeled template (with or without primer) is at fixed concentration of 10 nM. Primase concentrations range from 0 to 250 nM. The experiments in A and B were performed in triplicate; the error bars are the standard deviation. (C) Experimental design of the primase assay used to test the 5′ end effect on primer length determination. (D and E) Primer extension by wild-type primase, PriSL or PriSLXR72A. Note that the products run differently for 5′OH primer and 5′ monophosphate or triphosphate primers because of the charge difference. *The 9-nt product position for each primer is indicated. **PriSL concentration is 500 nM in this reaction.