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Antoine M. Van Oijen - One of the best experts on this subject based on the ideXlab platform.
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Replisome bypass of a protein based r loop block by pif1
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Grant D Schauer, Jacob S. Lewis, Lisanne M. Spenkelink, Antoine M. Van Oijen, Olga Yurieva, Stefan H MuellerAbstract: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 reconstituted Saccharomyces cerevisiae Replisome 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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a primase induced conformational switch controls the stability of the bacterial Replisome
Molecular Cell, 2020Co-Authors: Jacob S. Lewis, Slobodan Jergic, Nicholas E. Dixon, Enrico Monachino, Valerie L Oshea, James M Berger, Antoine M. Van OijenAbstract:Summary Recent studies of bacterial DNA replication have led to a picture of the Replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the Replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the Replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome
Nucleic acids research, 2019Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome.
2018Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:ABSTRACT Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events, and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here we visualise how an in vitro reconstituted E. coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualising SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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single molecule visualization of saccharomyces cerevisiae leading strand synthesis reveals dynamic interaction between mtc and the Replisome
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jacob S. Lewis, Mike Odonnell, Lisanne M. Spenkelink, Grant D Schauer, Flynn R Hill, Roxanna E Georgescu, Antoine M. Van OijenAbstract: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 e 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.
Mike Odonnell - One of the best experts on this subject based on the ideXlab platform.
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single molecule visualization of saccharomyces cerevisiae leading strand synthesis reveals dynamic interaction between mtc and the Replisome
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jacob S. Lewis, Mike Odonnell, Lisanne M. Spenkelink, Grant D Schauer, Flynn R Hill, Roxanna E Georgescu, Antoine M. Van OijenAbstract: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 e 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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the eukaryotic Replisome goes under the microscope
Current Biology, 2016Co-Authors: Mike OdonnellAbstract:The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic Replisome components include the Mcm2-7 complex, the CMG helicase, DNA polymerases, a Ctf4 trimer hub and the first look at a core Replisome of 20 different proteins containing the helicase, primase, leading polymerase and a lagging strand polymerase. The eukaryotic core Replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below. Additionally, structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the Replisome in handling nucleosomes, which are important to DNA organization and gene regulation. This review provides a summary of some of the many recent advances in the structure of the eukaryotic Replisome.
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the architecture of a eukaryotic Replisome
Nature Structural & Molecular Biology, 2015Co-Authors: Jingchuan Sun, Roxana E Georgescu, Mike Odonnell, Yi Shi, Zuanning Yuan, Brian T ChaitAbstract:At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae Replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic Replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical Replisome studies.
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reconstitution of a eukaryotic Replisome reveals suppression mechanisms that define leading lagging strand operation
eLife, 2015Co-Authors: Roxana E Georgescu, Nina Y Yao, Jeff Finkelstein, Lance D Langston, Grant D Schauer, Olga Yurieva, Dan Zhang, Mike OdonnellAbstract:Cells must replicate their DNA before they divide so that the newly formed cells can each receive a copy of the same genetic material. DNA replication requires complex molecular machinery called a Replisome, which comprises multiple proteins, enzymes, and other molecules. First, an enzyme called a helicase starts to unwind the double-stranded DNA into two single strands. This process continues while other enzymes, called polymerases, use the exposed single strands as templates to make complementary new strands of DNA. One of these new strands is built continuously and called the ‘leading strand’. The other newly forming strand—the ‘lagging strand’—is made in the opposite direction, as a series of short fragments that are later joined together. The Replisomes in bacterial cells have been well studied, but many researchers are investigating the composition of the Replisome in animals, plants, and fungi (collectively called eukaryotes). Now, Georgescu et al. have essentially rebuilt a eukaryotic Replisome from 31 different proteins in a test tube and confirmed that it can make both leading and lagging DNA strands—just like in a normal cell. Further experiments revealed that the polymerase enzyme that operates on the leading strand cannot work on the lagging strand and vice versa. This exclusivity is unique to eukaryotic DNA replication, as bacterial polymerases can use either DNA strand as a template. Georgescu et al. then found that the eukaryotic polymerases are actively prevented from copying the ‘wrong’ strand of DNA and suggest that the helicase enzyme that unwinds the DNA might be behind this activity. Important future studies must now address how the Replisome deals with obstacles created by certain DNA-binding proteins and damaged DNA and how it interfaces with the molecules that control cell division and DNA repair.
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reca acts as a switch to regulate polymerase occupancy in a moving replication fork
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: C Indiani, Meghna Patel, Myron F Goodman, Mike OdonnellAbstract:Abstract This report discovers a role of Escherichia coli RecA, the cellular recombinase, in directing the action of several DNA polymerases at the replication fork. Bulk chromosome replication is performed by DNA polymerase (Pol) III. However, E. coli contains translesion synthesis (TLS) Pols II, IV, and V that also function with the helicase, primase, and sliding clamp in the Replisome. Surprisingly, we find that RecA specifically activates Replisomes that contain TLS Pols. In sharp contrast, RecA severely inhibits the Pol III Replisome. Given the opposite effects of RecA on Pol III and TLS Replisomes, we propose that RecA acts as a switch to regulate the occupancy of polymerases within a moving Replisome.
Jacob S. Lewis - One of the best experts on this subject based on the ideXlab platform.
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Replisome bypass of a protein based r loop block by pif1
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Grant D Schauer, Jacob S. Lewis, Lisanne M. Spenkelink, Antoine M. Van Oijen, Olga Yurieva, Stefan H MuellerAbstract: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 reconstituted Saccharomyces cerevisiae Replisome 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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a primase induced conformational switch controls the stability of the bacterial Replisome
Molecular Cell, 2020Co-Authors: Jacob S. Lewis, Slobodan Jergic, Nicholas E. Dixon, Enrico Monachino, Valerie L Oshea, James M Berger, Antoine M. Van OijenAbstract:Summary Recent studies of bacterial DNA replication have led to a picture of the Replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the Replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the Replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
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single molecule imaging of eukaryotic Replisomes reveals compositional plasticity
bioRxiv, 2019Co-Authors: Jacob S. Lewis, Lisanne M. Spenkelink, Grant D Schauer, Olga Yurieva, Varsha Natarajan, Gurleen KaurAbstract:Duplication of the chromosomal DNA prior to cell division is performed by the Replisome, a multi-protein complex that coordinates a large number of enzymatic activities. The eukaryotic Replisome couples unwinding by the replicative DNA helicase (CMG) with the synthesis activity of DNA polymerases , {delta}, and {varepsilon} to simultaneously duplicate the leading and lagging strands. The ensemble-averaging nature of biochemical experiments obscures details on kinetics and stoichiometry of multi-protein complexes and makes it challenging to determine the exact composition of the Replisome during replication and whether protein components might dynamically be exchanged. Here we describe fluorescence imaging of the eukaryotic Replisome reconstituted from purified proteins and visualised at the single-molecule level while replicating DNA. We find that the two processive replicative DNA polymerases Pol {varepsilon} and Pol {delta} exchange stochastically and in a concentration-dependent manner, suggesting that the interactions within the Replisome are optimised to balance stability with plasticity. We further show that Pol {delta} synthesizes multiple Okazaki fragments while remaining attached to the Replisome, facilitated by interaction with Pol .
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome
Nucleic acids research, 2019Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome.
2018Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:ABSTRACT Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events, and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here we visualise how an in vitro reconstituted E. coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualising SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
Lisanne M. Spenkelink - One of the best experts on this subject based on the ideXlab platform.
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Replisome bypass of a protein based r loop block by pif1
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Grant D Schauer, Jacob S. Lewis, Lisanne M. Spenkelink, Antoine M. Van Oijen, Olga Yurieva, Stefan H MuellerAbstract: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 reconstituted Saccharomyces cerevisiae Replisome 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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single molecule imaging of eukaryotic Replisomes reveals compositional plasticity
bioRxiv, 2019Co-Authors: Jacob S. Lewis, Lisanne M. Spenkelink, Grant D Schauer, Olga Yurieva, Varsha Natarajan, Gurleen KaurAbstract:Duplication of the chromosomal DNA prior to cell division is performed by the Replisome, a multi-protein complex that coordinates a large number of enzymatic activities. The eukaryotic Replisome couples unwinding by the replicative DNA helicase (CMG) with the synthesis activity of DNA polymerases , {delta}, and {varepsilon} to simultaneously duplicate the leading and lagging strands. The ensemble-averaging nature of biochemical experiments obscures details on kinetics and stoichiometry of multi-protein complexes and makes it challenging to determine the exact composition of the Replisome during replication and whether protein components might dynamically be exchanged. Here we describe fluorescence imaging of the eukaryotic Replisome reconstituted from purified proteins and visualised at the single-molecule level while replicating DNA. We find that the two processive replicative DNA polymerases Pol {varepsilon} and Pol {delta} exchange stochastically and in a concentration-dependent manner, suggesting that the interactions within the Replisome are optimised to balance stability with plasticity. We further show that Pol {delta} synthesizes multiple Okazaki fragments while remaining attached to the Replisome, facilitated by interaction with Pol .
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome
Nucleic acids research, 2019Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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Recycling of single-stranded DNA-binding protein by the bacterial Replisome.
2018Co-Authors: Lisanne M. Spenkelink, Jacob S. Lewis, Slobodan Jergic, Andrew Robinson, Nicholas E. Dixon, Antoine M. Van OijenAbstract:ABSTRACT Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events, and playing many other regulatory roles within the Replisome. Recent developments in single-molecule approaches have led to a revised picture of the Replisome that is much more complex in how it retains or recycles protein components. Here we visualise how an in vitro reconstituted E. coli Replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualising SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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single molecule visualization of saccharomyces cerevisiae leading strand synthesis reveals dynamic interaction between mtc and the Replisome
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jacob S. Lewis, Mike Odonnell, Lisanne M. Spenkelink, Grant D Schauer, Flynn R Hill, Roxanna E Georgescu, Antoine M. Van OijenAbstract: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 e 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.
Joseph J. Loparo - One of the best experts on this subject based on the ideXlab platform.
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compartmentalization of the replication fork by single stranded dna binding protein regulates translesion synthesis
bioRxiv, 2020Co-Authors: Seungwoo Chang, Luisa Laureti, Vincent Pagès, Elizabeth S Thrall, Joseph J. LoparoAbstract:DNA replication is mediated by the coordinated actions of multiple enzymes within Replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving Replisomes, the remodeling of ssDNA binding protein (SSB) upon Replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.
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a gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion stalled Replisomes
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Seungwoo Chang, Slobodan Jergic, Nicholas E. Dixon, Karel Naiman, Elizabeth S Thrall, James E Kath, Robert P P Fuchs, Joseph J. LoparoAbstract: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 the Escherichia coli Replisome 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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a gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion stalled Replisomes
bioRxiv, 2019Co-Authors: Seungwoo Chang, Slobodan Jergic, Nicholas E. Dixon, Karel Naiman, Elizabeth S Thrall, James E Kath, Robert P P Fuchs, Joseph J. LoparoAbstract:Abstract 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 the bacterial Replisome 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 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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Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis
2015Co-Authors: Samir M. Hamdan, Joseph J. Loparo, Masateru Takahashi, Charles C. Richardson, Antoine M. Van OijenAbstract:In all organisms, the protein machinery responsible for the replication of DNA, the Replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments1,2. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki-fragment synthesis3. Here, we employ single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual Replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the Replisome after the synthesis of every Okazaki fragment
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simultaneous single molecule measurements of phage t7 Replisome composition and function reveal the mechanism of polymerase exchange
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Joseph J. Loparo, C Richardson, Arkadiusz W Kulczyk, Antoine M. Van OijenAbstract:A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage Replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing Replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the Replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.
