Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes
| Autor: | Charles C. Richardson, Karl E. Duderstadt, Antoine M. van Oijen, Sarah A. Stratmann, Arkadiusz W. Kulczyk, Hylkje Geertsema, Christiaan M. Punter |
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| Přispěvatelé: | Molecular Biophysics, Groningen Biomolecular Sciences and Biotechnology |
| Rok vydání: | 2016 |
| Předmět: |
DNA Replication
MECHANISM 0301 basic medicine PROTEINS COLI DNA-REPLICATION PRIMER SYNTHESIS Priming (immunology) DNA Primase Biology Time-Lapse Imaging 03 medical and health sciences chemistry.chemical_compound Gene duplication MOLECULE Lagging Molecular Biology PRIMASE Polymerase Genetics POLYMERASE DNA replication DNA Cell Biology EUKARYOTIC REPLICATION FORK Single Molecule Imaging Kinetics 030104 developmental biology chemistry ESCHERICHIA-COLI DNA Viral biology.protein Biophysics Replisome Primase BACTERIOPHAGE T7 |
| Zdroj: | Molecular Cell, 64(6), 1035-1047. CELL PRESS |
| ISSN: | 1097-2765 |
| DOI: | 10.1016/j.molcel.2016.10.028 |
| Popis: | 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 multi-dimensional 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. |
| Databáze: | OpenAIRE |
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