DNA replication dynamics: cooperative catalysis by the helicase‐primase and the DNA polymerase

DNA replication is coordinated by the action of a number of enzymatic activities. The ring‐shaped replicative helicase uses the energy of NTP hydrolyze to separate the DNA strands and two DNA polymerase molecules working closely with the helicase copy each of the strands in the 5′‐3′ direction. Due to the opposite polarity of the DNA strands, the leading‐strand DNA is copied in a continuous manner while the lagging‐strand DNA is copied in a discontinuous manner through Okazaki fragments. It is not understood how the two strands are synthesized with the same net rate. Using T7 replication proteins and approaches such as transient‐state kinetics and single molecule FRET, we find that RNA primers to make Okazaki fragments can be made ahead of time. However, during the time of primer synthesis, DNA replication continues un‐interrupted, evident from the formation of a ‘priming loop’. Therefore, the lagging polymerase still needs to make up the time for primer pick‐up and initiation of Okazaki fragment synthesis. Our kinetic studies show that the lagging‐strand polymerase copies the DNA at a faster rate than the leading‐strand replisome (polymerase‐helicase) that is limited by the speed of the helicase. Based on our studies, we propose that the differential rates of synthesis of the two strands and making primers ahead of time allows the two strands to be copied with the same net rate.