Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale

DNA polymerase delta (Pol delta) is responsible for elongation and maturation of Okazaki fragments. Pol 8 and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol 8 catalytic rate by >30-fold. When Pol delta invaded double-stranded RNA-DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10-20% that of the preceding nucleotide. Thus, the nascent flap acts as a progressive molecular brake on the polymerase, and consequently FEN1 cuts predominantly single-nucleotide flaps. Kinetic and enzyme trapping experiments support a model in which a stable PCNA-DNA-Pol delta-FEN1 complex moves processively through iterative steps of nick translation, ultimately completely removing primer RNA. Finally, whereas elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.