Resolving individual steps of Okazaki fragment maturation at msec time-scale

DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and flap-endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We have studied this process with the Saccharomyces cerevisiae machinery at msec resolution. During elongation, PCNA increased the catalytic rate of Pol δ by >30-fold. When Pol δ invaded double-stranded RNA-DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10–20% 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 δ-FEN1 complex moves processively through the iterative steps of nick translation in order to completely remove primer RNA. Finally, while elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.