scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

2021 ◽  
Author(s):  
Allison W. McClure ◽  
John F.X. Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Allison McClure ◽  
John Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Novel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation

2005 ◽  
Vol 25 (6) ◽  
pp. 2463-2474 ◽  
Author(s):  
Kyung Kim ◽  
Diana D. Dimitrova ◽  
Kristine M. Carta ◽  
Anjana Saxena ◽  
Mariza Daras ◽  
...  
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
Protein A ◽  
Resonance Energy ◽  
Genotoxic Stress ◽  
Replication Protein A ◽  
Replication Protein ◽  
Chromosomal Dna

ABSTRACT Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

scholarly journals Werner Syndrome Protein and DNA Replication

2018 ◽  
Vol 19 (11) ◽  
pp. 3442 ◽  
Author(s):  
Shibani Mukherjee ◽  
Debapriya Sinha ◽  
Souparno Bhattacharya ◽  
Kalayarasan Srinivasan ◽  
Salim Abdisalaam ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ataxia Telangiectasia ◽  
Replication Fork ◽  
Genome Stability ◽  
Werner Syndrome ◽  
Genotoxic Stress ◽  
Premature Aging ◽  
Replication Forks ◽  
Werner Syndrome Protein ◽  
Syndrome Protein

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3′ to 5′ helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN’s contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN’s molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

scholarly journals LncRNA CARMN overexpression promotes prognosis and chemosensitivity of triple negative breast cancer via acting as miR143-3p host gene and inhibiting DNA replication

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Xiaonan Sheng ◽  
Huijuan Dai ◽  
Yueyao Du ◽  
Jing Peng ◽  
Rui Sha ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Replication ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Replication Initiation ◽  
Host Gene ◽  
Initiation Factor ◽  
Rna Seq

Abstract Background Triple negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and lack of effective treatment target. Here we screened differentially expressed lncRNAs through bioinformatics analysis and identified CARMN as a downregulated lncRNA which is lowest expressed in TNBC. We aimed to identify the potential role and molecular mechanisms of CARMN in TNBC. Methods Predictive value of CARMN was explored in breast cancer cohorts. TNBC cell lines with CARMN overexpression or CARMN silence and were used for in vitro and in vivo experiments. RNA-seq of CARMN overexpressed cells was performed for exploring downstream of CARMN. Results CARMN is downregulated at different phase of malignant transformation of breast tissue. CARMN can predict both better prognosis and higher response rate of cisplatin-based neoadjuvant chemotherapy in breast cancer. A nomogram is built to predict cisplatin-based chemotherapy response in breast cancer. Through in vitro and in vivo studies, we confirmed CARMN can also inhibit tumorigenesis and enhance sensitivity to cisplatin in TNBC cells. RNA-seq and further experiments revealed CARMN can inhibit DNA replication. MCM5, an important DNA replication initiation factor, is the most downregulated gene in DNA replication pathway following CARMN overexpression. We confirmed CARMN can produce miR143-3p from its exon5 which is DROSHA and DICER dependent, resulting binding and decrease of MCM5. Moreover, suppressing miR143-3p can weaken function of CARMN in suppressing tumorigenesis and promoting chemosensitivity. Conclusions Our results indicated lncRNA CARMN is a predictive biomarker of better prognosis and enhanced cisplatin sensitivity in TNBC. CARMN is the host gene of miR143-3p which downregulates MCM5, causing inhibited DNA replication.

scholarly journals Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase—Pol ε Interaction

2020 ◽  
Vol 21 (24) ◽  
pp. 9484
Author(s):  
Milena Denkiewicz-Kruk ◽  
Malgorzata Jedrychowska ◽  
Shizuko Endo ◽  
Hiroyuki Araki ◽  
Piotr Jonczyk ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Synthetic Lethality ◽  
Replication Initiation ◽  
Recombination Rates ◽  
Dna Polymerase Epsilon ◽  
Dna Replication Initiation ◽  
Leading Strand

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kelsey S. Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant D. Schauer ◽  
Stefan H. Mueller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Molecular Machines ◽  
Enzymatic Activities ◽  
Replication Forks ◽  
Dna Substrates ◽  
Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Replication Restart in Bacteria

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Bénédicte Michel ◽  
Steven J. Sandler
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Strand Break ◽  
Replication Initiation ◽  
Replication Forks ◽  
Independent Manner ◽  
Close Link ◽  
Replication Restart

ABSTRACT In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.

scholarly journals Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe

2008 ◽  
Vol 28 (22) ◽  
pp. 6844-6857 ◽  
Author(s):  
Subhrajit Biswas ◽  
Deepak Bastia
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Replication Fork ◽  
Intrinsic Property ◽  
Dimerization Domain ◽  
Mammalian Counterpart ◽  
Chromosome Iii ◽  
Necessary And Sufficient ◽  
Replication Fork Arrest

ABSTRACT Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the “sweepase” Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

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