Journal articles: 'Replisome'

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Galarreta, Antonio, Pablo Valledor, Oscar Fernandez-Capetillo, and Emilio Lecona. "Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1." International Journal of Molecular Sciences 22, no. 16 (August 16, 2021): 8796. http://dx.doi.org/10.3390/ijms22168796.

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Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.

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Priego Moreno, Sara, Rebecca M. Jones, Divyasree Poovathumkadavil, Shaun Scaramuzza, and Agnieszka Gambus. "Mitotic replisome disassembly depends on TRAIP ubiquitin ligase activity." Life Science Alliance 2, no. 2 (April 2019): e201900390. http://dx.doi.org/10.26508/lsa.201900390.

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We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a “backup” for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.

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Li, Huilin, Nina Y. Yao, and Michael E. O'Donnell. "Anatomy of a twin DNA replication factory." Biochemical Society Transactions 48, no. 6 (December 10, 2020): 2769–78. http://dx.doi.org/10.1042/bst20200640.

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The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

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Kapadia, Nitin, and Rodrigo Reyes-Lamothe. "A quest for coordination among activities at the replisome." Biochemical Society Transactions 47, no. 4 (August 8, 2019): 1067–75. http://dx.doi.org/10.1042/bst20180402.

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Abstract Faithful DNA replication is required for transmission of the genetic material across generations. The basic mechanisms underlying this process are shared among all organisms: progressive unwinding of the long double-stranded DNA; synthesis of RNA primers; and synthesis of a new DNA chain. These activities are invariably performed by a multi-component machine called the replisome. A detailed description of this molecular machine has been achieved in prokaryotes and phages, with the replication processes in eukaryotes being comparatively less known. However, recent breakthroughs in the in vitro reconstitution of eukaryotic replisomes have resulted in valuable insight into their functions and mechanisms. In conjunction with the developments in eukaryotic replication, an emerging overall view of replisomes as dynamic protein ensembles is coming into fruition. The purpose of this review is to provide an overview of the recent insights into the dynamic nature of the bacterial replisome, revealed through single-molecule techniques, and to describe some aspects of the eukaryotic replisome under this framework. We primarily focus on Escherichia coli and Saccharomyces cerevisiae (budding yeast), since a significant amount of literature is available for these two model organisms. We end with a description of the methods of live-cell fluorescence microscopy for the characterization of replisome dynamics.

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Chang, Seungwoo, Karel Naiman, Elizabeth S. Thrall, James E. Kath, Slobodan Jergic, Nicholas E. Dixon, Robert P. Fuchs, and Joseph J. Loparo. "A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes." Proceedings of the National Academy of Sciences 116, no. 51 (December 3, 2019): 25591–601. http://dx.doi.org/10.1073/pnas.1914485116.

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DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within theEscherichia colireplisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

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Jenkyn-Bedford, Michael, Morgan L. Jones, Yasemin Baris, Karim P. M. Labib, Giuseppe Cannone, Joseph T. P. Yeeles, and Tom D. Deegan. "A conserved mechanism for regulating replisome disassembly in eukaryotes." Nature 600, no. 7890 (October 26, 2021): 743–47. http://dx.doi.org/10.1038/s41586-021-04145-3.

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AbstractReplisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase1–3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4–7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8–10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome–E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR–MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

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Lewis, Jacob S., Lisanne M. Spenkelink, Grant D. Schauer, Flynn R. Hill, Roxanna E. Georgescu, Michael E. O’Donnell, and Antoine M. van Oijen. "Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): 10630–35. http://dx.doi.org/10.1073/pnas.1711291114.

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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 ε 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.

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Wang, Jue D., Megan E. Rokop, Melanie M. Barker, Nathaniel R. Hanson, and Alan D. Grossman. "Multicopy Plasmids Affect Replisome Positioning in Bacillus subtilis." Journal of Bacteriology 186, no. 21 (November 1, 2004): 7084–90. http://dx.doi.org/10.1128/jb.186.21.7084-7090.2004.

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ABSTRACT The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

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Nye, Dillon B., and Nathan A. Tanner. "Chimeric DNA byproducts in strand displacement amplification using the T7 replisome." PLOS ONE 17, no. 9 (September 19, 2022): e0273979. http://dx.doi.org/10.1371/journal.pone.0273979.

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Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.

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Attali, Ilan, Michael R. Botchan, and James M. Berger. "Structural Mechanisms for Replicating DNA in Eukaryotes." Annual Review of Biochemistry 90, no. 1 (June 20, 2021): 77–106. http://dx.doi.org/10.1146/annurev-biochem-090120-125407.

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The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein–nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.

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Guarino Almeida, Estrella, Xavier Renaudin, and Ashok R. Venkitaraman. "A kinase-independent function for AURORA-A in replisome assembly during DNA replication initiation." Nucleic Acids Research 48, no. 14 (July 11, 2020): 7844–55. http://dx.doi.org/10.1093/nar/gkaa570.

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Abstract The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.

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Syeda, Aisha H., Adam J. M. Wollman, Alex L. Hargreaves, Jamieson A. L. Howard, Jan-Gert Brüning, Peter McGlynn, and Mark C. Leake. "Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome." Nucleic Acids Research 47, no. 12 (April 27, 2019): 6287–98. http://dx.doi.org/10.1093/nar/gkz298.

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Abstract DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

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Hadjicharalambous, Andreas, Alex J. Whale, Geylani Can, J. Mark Skehel, Jonathan M. Houseley, and Philip Zegerman. "Checkpoint kinase interaction with DNA polymerase alpha regulates replication progression during stress." Wellcome Open Research 8 (July 26, 2023): 327. http://dx.doi.org/10.12688/wellcomeopenres.19617.1.

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Background: In eukaryotes, replication stress activates a checkpoint response, which facilitates genome duplication by stabilising the replisome. How the checkpoint kinases regulate the replisome remains poorly understood. The aim of this study is to identify new targets of checkpoint kinases within the replisome during replication stress. Methods: Here we use an unbiased biotin proximity-ligation approach in Saccharomyces cerevisiae to identify new interactors and substrates of the checkpoint kinase Rad53 in vivo. Results: From this screen, we identified the replication initiation factor Sld7 as a Rad53 substrate, and Pol1, the catalytic subunit of polymerase a, as a Rad53-interactor. We showed that CDK phosphorylation of Pol1 mediates its interaction with Rad53. Combined with other interactions between Rad53 and the replisome, this Rad53-Pol1 interaction is important for viability and replisome progression during replication stress. Conclusions: Together, we explain how the interactions of Rad53 with the replisome are controlled by both replication stress and the cell cycle, and why these interactions might be important for coordinating the stabilisation of both the leading and lagging strand machineries.

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Liao, Yi, Jeremy W. Schroeder, Burke Gao, Lyle A. Simmons, and Julie S. Biteen. "Single-molecule motions and interactions in live cells reveal target search dynamics in mismatch repair." Proceedings of the National Academy of Sciences 112, no. 50 (November 2, 2015): E6898—E6906. http://dx.doi.org/10.1073/pnas.1507386112.

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MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.

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Bell, Stephen P. "Terminating the replisome." Science 346, no. 6208 (October 23, 2014): 418–19. http://dx.doi.org/10.1126/science.1261245.

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Yao, Nina Y., and Mike O'Donnell. "SnapShot: The Replisome." Cell 141, no. 6 (June 2010): 1088–1088. http://dx.doi.org/10.1016/j.cell.2010.05.042.

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Lerner, Leticia Koch, and Julian E. Sale. "Replication of G Quadruplex DNA." Genes 10, no. 2 (January 29, 2019): 95. http://dx.doi.org/10.3390/genes10020095.

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A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

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Duckworth, Alexander T., Tricia A. Windgassen, and James L. Keck. "Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity." PLOS ONE 16, no. 7 (July 30, 2021): e0255409. http://dx.doi.org/10.1371/journal.pone.0255409.

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DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

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Benkovic, Stephen J., Ann M. Valentine, and Frank Salinas. "Replisome-Mediated DNA Replication." Annual Review of Biochemistry 70, no. 1 (June 2001): 181–208. http://dx.doi.org/10.1146/annurev.biochem.70.1.181.

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Moses, Robb E., Anne Byford, and James A. Hejna. "Replisome pausing in mutagenesis." Chromosoma 102, S1 (December 1992): S157—S160. http://dx.doi.org/10.1007/bf02451801.

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Chai, Tiancong, Céline Terrettaz, and Justine Collier. "Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus." Nucleic Acids Research 49, no. 6 (March 2, 2021): 3308–21. http://dx.doi.org/10.1093/nar/gkab112.

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Abstract The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.

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Zhou, Haixia, Manal S. Zaher, Johannes C. Walter, and Alan Brown. "Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates." Nucleic Acids Research 49, no. 22 (December 1, 2021): 13194–206. http://dx.doi.org/10.1093/nar/gkab1174.

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Abstract When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1’s catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

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Moreno, Sara Priego, and Agnieszka Gambus. "Mechanisms of eukaryotic replisome disassembly." Biochemical Society Transactions 48, no. 3 (June 3, 2020): 823–36. http://dx.doi.org/10.1042/bst20190363.

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DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

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Brüning, Jan-Gert, and Kenneth J. Marians. "Replisome bypass of transcription complexes and R-loops." Nucleic Acids Research 48, no. 18 (September 14, 2020): 10353–67. http://dx.doi.org/10.1093/nar/gkaa741.

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Abstract The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

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Somyajit, Kumar, Rajat Gupta, Hana Sedlackova, Kai John Neelsen, Fena Ochs, Maj-Britt Rask, Chunaram Choudhary, and Jiri Lukas. "Redox-sensitive alteration of replisome architecture safeguards genome integrity." Science 358, no. 6364 (November 9, 2017): 797–802. http://dx.doi.org/10.1126/science.aao3172.

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DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)–generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.

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Marians, Kenneth J. "Understanding how the replisome works." Nature Structural & Molecular Biology 15, no. 2 (February 2008): 125–27. http://dx.doi.org/10.1038/nsmb0208-125.

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Mao, Steve. "Structures of the simplest replisome." Science 363, no. 6429 (February 21, 2019): 831.12–833. http://dx.doi.org/10.1126/science.363.6429.831-l.

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Spinks, Richard R., Lisanne M. Spenkelink, Sarah A. Stratmann, Zhi-Qiang Xu, N. Patrick J. Stamford, Susan E. Brown, Nicholas E. Dixon, Slobodan Jergic, and Antoine M. van Oijen. "DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies." Nucleic Acids Research 49, no. 12 (June 17, 2021): 6804–16. http://dx.doi.org/10.1093/nar/gkab493.

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Abstract In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.

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Kilkenny, Mairi L., Aline C. Simon, Jack Mainwaring, David Wirthensohn, Sandro Holzer, and Luca Pellegrini. "The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box." Open Biology 7, no. 11 (November 2017): 170217. http://dx.doi.org/10.1098/rsob.170217.

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A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.

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Muylaert, Isabella, Ka-Wei Tang, and Per Elias. "Replication and Recombination of Herpes Simplex Virus DNA." Journal of Biological Chemistry 286, no. 18 (March 1, 2011): 15619–24. http://dx.doi.org/10.1074/jbc.r111.233981.

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Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.

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Bellani, Marina A., Althaf Shaik, Ishani Majumdar, Chen Ling, and Michael M. Seidman. "The Response of the Replication Apparatus to Leading Template Strand Blocks." Cells 12, no. 22 (November 11, 2023): 2607. http://dx.doi.org/10.3390/cells12222607.

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Duplication of the genome requires the replication apparatus to overcome a variety of impediments, including covalent DNA adducts, the most challenging of which is on the leading template strand. Replisomes consist of two functional units, a helicase to unwind DNA and polymerases to synthesize it. The helicase is a multi-protein complex that encircles the leading template strand and makes the first contact with a leading strand adduct. The size of the channel in the helicase would appear to preclude transit by large adducts such as DNA: protein complexes (DPC). Here we discuss some of the extensively studied pathways that support replication restart after replisome encounters with leading template strand adducts. We also call attention to recent work that highlights the tolerance of the helicase for adducts ostensibly too large to pass through the central channel.

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Claussin, Clémence, Jacob Vazquez, and Iestyn Whitehouse. "Single-molecule mapping of replisome progression." Molecular Cell 82, no. 7 (April 2022): 1372–82. http://dx.doi.org/10.1016/j.molcel.2022.02.010.

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Sun, Jingchuan, Yi Shi, Roxana E. Georgescu, Zuanning Yuan, Brian T. Chait, Huilin Li, and Michael E. O'Donnell. "The architecture of a eukaryotic replisome." Nature Structural & Molecular Biology 22, no. 12 (November 2, 2015): 976–82. http://dx.doi.org/10.1038/nsmb.3113.

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van Oijen, Antoine M., and Joseph J. Loparo. "Single-Molecule Studies of the Replisome." Annual Review of Biophysics 39, no. 1 (April 2010): 429–48. http://dx.doi.org/10.1146/annurev.biophys.093008.131327.

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Bates, David. "The bacterial replisome: back on track?" Molecular Microbiology 69, no. 6 (September 2008): 1341–48. http://dx.doi.org/10.1111/j.1365-2958.2008.06378.x.

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Shamoo, Yousif, and Thomas A. Steitz. "Building a Replisome from Interacting Pieces." Cell 99, no. 2 (October 1999): 155–66. http://dx.doi.org/10.1016/s0092-8674(00)81647-5.

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Sutani, Takashi, and Katsuhiko Shirahige. "Attaching Accessory Devices to the Replisome." Molecular Cell 63, no. 3 (August 2016): 347–48. http://dx.doi.org/10.1016/j.molcel.2016.07.017.

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O'Donnell, Mike. "Replisome Architecture and Dynamics inEscherichia coli." Journal of Biological Chemistry 281, no. 16 (January 18, 2006): 10653–56. http://dx.doi.org/10.1074/jbc.r500028200.

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Villa‐Hernández, Sara, and Rodrigo Bermejo. "Replisome‐Cohesin Interfacing: A Molecular Perspective." BioEssays 40, no. 10 (August 14, 2018): 1800109. http://dx.doi.org/10.1002/bies.201800109.

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Wang, Yilin, Kathryn S. Brady, Benjamin P. Caiello, Stephanie M. Ackerson, and Jason A. Stewart. "Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association." Life Science Alliance 2, no. 2 (April 2019): e201800270. http://dx.doi.org/10.26508/lsa.201800270.

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Human CTC1-STN1-TEN1 (CST) is an RPA-like single-stranded DNA-binding protein that interacts with DNA polymerase α-primase (pol α) and functions in telomere replication. Previous studies suggest that CST also promotes replication restart after fork stalling. However, the precise role of CST in genome-wide replication remains unclear. In this study, we sought to understand whether CST alters origin licensing and activation. Replication origins are licensed by loading of the minichromosome maintenance 2–7 (MCM) complex in G1 followed by replisome assembly and origin firing in S-phase. We find that CST directly interacts with the MCM complex and disrupts binding of CDT1 to MCM, leading to decreased origin licensing. We also show that CST enhances replisome assembly by promoting AND-1/pol α chromatin association. Moreover, these interactions are not dependent on exogenous replication stress, suggesting that CST acts as a specialized replication factor during normal replication. Overall, our findings implicate CST as a novel regulator of origin licensing and replisome assembly/fork progression through interactions with MCM, AND-1, and pol α.

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Schauer, Grant D., Lisanne M. Spenkelink, Jacob S. Lewis, Olga Yurieva, Stefan H. Mueller, Antoine M. van Oijen, and Michael E. O’Donnell. "Replisome bypass of a protein-based R-loop block by Pif1." Proceedings of the National Academy of Sciences 117, no. 48 (November 16, 2020): 30354–61. http://dx.doi.org/10.1073/pnas.2020189117.

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Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstitutedSaccharomyces cerevisiaereplisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

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Delagoutte, Emmanuelle, and Giuseppe Baldacci. "5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs." Molecular Biology International 2011 (August 10, 2011): 1–14. http://dx.doi.org/10.4061/2011/213824.

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Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs.

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van Schie, Janne J. M., and Job de Lange. "The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks." Cells 10, no. 12 (December 8, 2021): 3455. http://dx.doi.org/10.3390/cells10123455.

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The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

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Vrtis, Kyle B., James M. Dewar, Gheorghe Chistol, R. Alex Wu, Thomas G. W. Graham, and Johannes C. Walter. "Single-strand DNA breaks cause replisome disassembly." Molecular Cell 81, no. 6 (March 2021): 1309–18. http://dx.doi.org/10.1016/j.molcel.2020.12.039.

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D'Angiolella, Vincenzo, and Daniele Guardavaccaro. "Two paths to let the replisome go." Cell Death & Differentiation 24, no. 7 (May 19, 2017): 1140–41. http://dx.doi.org/10.1038/cdd.2017.75.

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Hamdan, Samir M., and Charles C. Richardson. "Motors, Switches, and Contacts in the Replisome." Annual Review of Biochemistry 78, no. 1 (June 2009): 205–43. http://dx.doi.org/10.1146/annurev.biochem.78.072407.103248.

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Gao, Yang, Yanxiang Cui, Tara Fox, Shiqiang Lin, Huaibin Wang, Natalia de Val, Z. Hong Zhou, and Wei Yang. "Structures and operating principles of the replisome." Science 363, no. 6429 (January 24, 2019): eaav7003. http://dx.doi.org/10.1126/science.aav7003.

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Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

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Schaeffer, Patrick, Madeleine Headlam, and Nicholas Dixon. "Protein – Protein Interactions in the Eubacterial Replisome." IUBMB Life (International Union of Biochemistry and Molecular Biology: Life) 57, no. 1 (January 1, 2005): 5–12. http://dx.doi.org/10.1080/15216540500058956.

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Baker, Tania A., and Stephen P. Bell. "Polymerases and the Replisome: Machines within Machines." Cell 92, no. 3 (February 1998): 295–305. http://dx.doi.org/10.1016/s0092-8674(00)80923-x.

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McInerney, Peter, Aaron Johnson, Francine Katz, and Mike O'Donnell. "Characterization of a Triple DNA Polymerase Replisome." Molecular Cell 27, no. 4 (August 2007): 527–38. http://dx.doi.org/10.1016/j.molcel.2007.06.019.

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Journal articles on the topic 'Replisome'

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