A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.
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http://dx.doi.org/10.1016/s1040-8428(03)00096-9 | DOI Listing |
Nature
September 2024
Laboratory for Developmental Epigenetics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan.
Faithful DNA replication is essential for genome integrity. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis. However, the regulation of DNA replication remains poorly understood in early mammalian embryos.
View Article and Find Full Text PDFNucleic Acids Res
September 2024
Department of Molecular and Cellular Biology, University of Geneva, 1211 Geneva 4, Switzerland.
DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1.
View Article and Find Full Text PDFNat Commun
July 2024
Dept. of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
In Escherichia coli, it is debated whether the two replisomes move independently along the two chromosome arms during replication or if they remain spatially confined. Here, we use high-throughput fluorescence microscopy to simultaneously determine the location and short-time-scale (1 s) movement of the replisome and a chromosomal locus throughout the cell cycle. The assay is performed for several loci.
View Article and Find Full Text PDFNucleic Acids Res
August 2024
Bioscience Program, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis.
View Article and Find Full Text PDFNature
April 2024
Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland.
An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair.
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