Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome
| Autor: | Flynn R. Hill, Grant D. Schauer, Roxanna E. Georgescu, Mike O'Donnell, Jacob S. Lewis, Antoine M. van Oijen, Lisanne M. Spenkelink |
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| Přispěvatelé: | Molecular Biophysics |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
CHROMATIN Saccharomyces cerevisiae Proteins DNA polymerase single-molecule biophysics Cell Cycle Proteins PROGRESSION Saccharomyces cerevisiae MRC1 DNA replication Pre-replication complex CMG ACTIVATION 03 medical and health sciences INITIATION 0302 clinical medicine Minichromosome maintenance TOF1 DNA Fungal Multidisciplinary DNA clamp biology Minichromosome Maintenance Proteins replisome ELONGATION MCM10 Helicase Biological Sciences CMG HELICASE Molecular biology Cell biology DNA-Binding Proteins 030104 developmental biology Multiprotein Complexes biology.protein Replisome DNA-REPLICATION FORKS 030217 neurology & neurosurgery |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America, 114(40), 10630-10635. NATL ACAD SCIENCES |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1711291114 |
| Popis: | The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol epsilon DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1-Tof1-Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication. |
| Databáze: | OpenAIRE |
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