Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation. [Display omitted] •Simultaneous imaging of leading- and lagging-strand synthesis by single replisomes•Most loop formation events on the lagging strand occur during primer synthesis•Polymerases are released from the replisome to complete Okazaki-fragment synthesis•Multiple pathways and exchange events underlie replisome coordination Duderstadt et al. report a multidimensional single-molecule approach to simultaneously observe DNA looping and leading-strand synthesis during replication. Working with the bacteriophage T7 replisome, they show that most DNA loops form only during priming to support several parallel pathways that ensure robust coordination of daughter-strand synthesis.