The faithful transmission of genetic information from one generation to the next is the most vital cellular function for the proliferation of life. Fundamental to this process is the duplication of chromosomes, which depends on many distinct enzymes within large protein complexes known as replisomes. These replication factories serve as organizational centers in which enzymatic events are ordered and coupled. Inside these vast structures chromosomal DNA is separated from bound factors and unwound to generate templates for the synthesis of daughter chromosomes. A myriad of short-lived intermediates and dynamic structures form during this process but few methods offer a direct glimpse of these events.
To study the dynamics of DNA replication we developed a multidimensional single-molecule imaging approach that provides a direct, real-time view of the timing of polymerase synthesis on the two daughter strands. To follow the dynamics within single replisomes, micron-sized beads were attached to single DNA molecules immobilized on microscope slides. A constant flow of liquid was then used to stretch the individual DNA molecules so that length changes, corresponding to DNA synthesis events, could be precisely followed by tracking the beads. By attaching two beads at different ends of each DNA molecule we were able to independently record the synthesis dynamics on each daughter strand and correlate them in time.
Figure 1: A multidimensional single-molecule imaging approach reveals the synthesis dynamics of single replisomes. Several reaction pathways ensure robust coordination and timing of daughter-strand synthesis.
This novel imaging approach allowed us to directly evaluate several competing models for replisome coordination providing new insight into a longstanding debate about why the synthesis of one daughter strand does not outpace that of the other. We discovered that most synthesis on one of the daughter strands, known as the lagging strand, is conducted by polymerases left in the wake of the DNA replication machinery. Single-molecule fluorescence imaging revealed that new polymerases rapidly bind to replace those left behind. This partial uncoupling of polymerase synthesis may be one mechanism used to ensure coordinated duplication of daughter chromosomes.
Figure 2: Structural model of a phage replisome in operation. Parental duplex DNA (gray) is unwound by a helicase (blue) which generates two single-stranded templates for the synthesis of new daughter strands (orange) by several DNA polymerases (green/yellow). In the model shown, one strand is copied continuously while a single-stranded loop bound by proteins (red) forms on the other prior to synthesis. Original image by Jacob Lewis, University of Wollongong, Australia.
Simultaneous imaging of daughter-strand synthesis provides a holistic view of different enzymatic events that occur during replication revealing that DNA looping and pausing events support the robust timing of synthesis. These results suggest far more plasticity in replisome operation than previously believed. This flexibility may be a byproduct of the complex acrobatics that occur within the replisome, or it may be required for specialized bypass pathways that allow replisomes to rapidly recognize and overcome obstacles encountered on parental chromosomes.
Reference
Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes. Duderstadt, K.E., Geertsema, H.J., Stratmann, S.A., Punter, C.M., Kulczyk, A.W., Richardson, C.C., van Oijen, A.M., 2016. Molecular Cell 64, 1035–1047. doi:10.1016/j.molcel.2016.10.028.
Other Reference
Stability versus exchange: a paradox in DNA replication. Åberg, C., Duderstadt, K.E., van Oijen, A.M., 2016. Nucleic Acids Research 44, 4846–4854. doi:10.1093/nar/gkw296.