Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast

2017 ◽  
pp. 227-238 ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Zhiguo Zhang
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Budding Yeast ◽  
Replication Forks ◽  
Specific Analysis

Single-molecule binding characterization of primosomal protein PriA involved in replication restart

2021 ◽  
Author(s):  
Tzu-Yu Lee ◽  
Yi-Ching Li ◽  
Min-Guan Lin ◽  
Chwan-Deng Hsiao ◽  
Hung-Wen Li
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Replication Forks ◽  
Bacterial Survival ◽  
Dna Damages ◽  
Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

scholarly journals Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

2021 ◽  
Author(s):  
Cory Haluska ◽  
Fengzhi Jin ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Replication Stress ◽  
S Phase ◽  
Viability Loss ◽  
Phosphatase 2A ◽  
Dna Replication Stress

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

scholarly journals Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

2019 ◽  
Author(s):  
Sujan Devbhandari ◽  
Dirk Remus
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Point Mutations ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Replication Checkpoint ◽  
Polymerase Domain ◽  
Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

scholarly journals CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tom D Deegan ◽  
Progya P Mukherjee ◽  
Ryo Fujisawa ◽  
Cristian Polo Rivera ◽  
Karim Labib
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
General Model ◽  
Replication Fork ◽  
Protein Complexes ◽  
Budding Yeast ◽  
Dna Structure ◽  
Replication Forks ◽  
Ubiquitylated Protein

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a ‘5-ubiquitin threshold’ that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.

scholarly journals The integrated stress response induces R-loops and hinders replication fork progression

2020 ◽  
Author(s):  
Josephine Ann Mun Yee Choo ◽  
Denise Schlösser ◽  
Valentina Manzini ◽  
Anna Magerhans ◽  
Matthias Dobbelstein
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Stress Response ◽  
Dna Synthesis ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Replication Forks ◽  
Integrated Stress Response ◽  
Loop Formation ◽  
Initiation Of Translation

ABSTRACTThe integrated stress response (ISR) allows cells to rapidly shut down most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within one hour, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or re-activate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops) which interfere with DNA replication. Histone depletion alone induces R-loops and compromises DNA replication. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses.SIGNIFICANCEThe integrated stress response has long been explored regarding its immediate impact on protein synthesis. Translational shutdown represents an indispensable mechanism to prevent the toxicity of misfolded proteins and virus infections. Our results indicate that the shutdown mechanisms reach far beyond translation and immediately interfere with DNA synthesis as well. ISR depletes cells of new histones which induce accumulation of DNA:RNA hybrids. The impairment of DNA replication in this context supports cell survival during stress.Our work provides a link between the ISR and another subject of active research, i. e. the regulatory network of DNA replication forks.Graphical Abstract

scholarly journals Phosphorylation of PNKP mediated by CDKs promotes end-processing of Okazaki fragments during DNA replication

2021 ◽  
Author(s):  
Kaima Tsukada ◽  
Rikiya Imamura ◽  
Kotaro Saikawa ◽  
Mizuki Saito ◽  
Naoya Kase ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
High Speed ◽  
Replication Forks ◽  
Cyclin Dependent Kinases ◽  
Okazaki Fragments ◽  
Dna Ligation ◽  
Polynucleotide Kinase ◽  
Dna Ends

Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′ phosphatase and 5′ kinase of DNA ends to promote DNA ligation. Here, we show that PNKP is involved in progression of DNA replication through end-processing of Okazaki fragments (OFs). Cyclin-dependent kinases (CDKs) regulate phosphorylation on threonine 118 (T118) of PNKP, and which phosphorylation allows it to be recruited to OFs. Loss of PNKP and T118 phosphorylation significantly increased unligated OFs and high-speed DNA synthesis in replication forks, suggesting that PNKP T118 phosphorylation is required for OFs ligation for its maturation. Furthermore, phosphatase-dead PNKP also exhibited an accumulation of unligated OFs and high-speed DNA synthesis. Overall, our data suggested that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to OFs and PNKP subsequently promotes end-processing for OFs maturation for stable cell proliferation.

scholarly journals Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

2008 ◽  
Vol 19 (11) ◽  
pp. 4956-4967 ◽  
Author(s):  
Hsiao-Chi Lo ◽  
Lihong Wan ◽  
Adam Rosebrock ◽  
Bruce Futcher ◽  
Nancy M. Hollingsworth
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Budding Yeast ◽  
Genetic Approach ◽  
Chemical Genetic ◽  
Meiotic Progression ◽  
Cdc7 Kinase ◽  
Yeast Meiosis ◽  
Meiosis I

In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis.

scholarly journals Mrc1 and Tof1 Regulate DNA Replication Forks in Different Ways during Normal S Phase

2007 ◽  
Vol 18 (10) ◽  
pp. 3894-3902 ◽  
Author(s):  
Ben Hodgson ◽  
Arturo Calzada ◽  
Karim Labib
Keyword(s):  
Dna Replication ◽  
Budding Yeast ◽  
Chromosome Replication ◽  
S Phase ◽  
Replication Forks ◽  
Normal Process ◽  
Checkpoint Kinases ◽  
Mcm Helicase ◽  
Rate Of Progression

The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1.

scholarly journals SCFDia2 regulates DNA replication forks during S-phase in budding yeast

2009 ◽  
Vol 28 (23) ◽  
pp. 3693-3705 ◽  
Author(s):  
Satoru Mimura ◽  
Makiko Komata ◽  
Tsutomu Kishi ◽  
Katsuhiko Shirahige ◽  
Takumi Kamura
Keyword(s):  
Dna Replication ◽  
Budding Yeast ◽  
S Phase ◽  
Replication Forks

The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase

2001 ◽  
Vol 114 (4) ◽  
pp. 747-750
Author(s):  
J. Malinsky ◽  
K. Koberna ◽  
D. Stanek ◽  
M. Masata ◽  
I. Votruba ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Hela Cells ◽  
Replication Fork ◽  
S Phase ◽  
Limiting Factor ◽  
Replication Forks ◽  
Replication Process ◽  
Average Speed ◽  
Rate Limiting

Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2′-deoxyribonucleoside 5′-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2′-deoxyribonucleoside 5′-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2′-deoxythymidine analogues 5-chloro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2′-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.

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