Nuclease dead Cas9 is a programmable roadblock for DNA replication

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.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

Scientific Reports ◽

10.1038/s41598-019-49837-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 10

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.

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

10.1101/2021.04.09.439171 ◽

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.

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

eLife ◽

10.7554/elife.69726 ◽

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.

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Delineation of the Ancestral Tus-Dependent Replication Fork Trap

10.20944/preprints202111.0539.v1 ◽

2021 ◽

Author(s):

Casey Toft ◽

Morgane Moreau ◽

Jiri Perutka ◽

Savitri Mandapati ◽

Peter Enyeart ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Replication Fork ◽

Wide Distribution ◽

Replication Forks ◽

Genome Wide ◽

Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

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Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

Author(s):

D. T. Gruszka ◽

S. Xie ◽

H. Kimura ◽

H. Yardimci

Keyword(s):

Dna Replication ◽

Real Time ◽

Single Molecule ◽

Replication Fork ◽

Epigenetic Inheritance ◽

Replication Forks ◽

Replication Fork Progression ◽

Egg Extracts ◽

Fork Stalling ◽

The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

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Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814512116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1251-1260 ◽

Cited By ~ 7

Author(s):

Glen E. Cronan ◽

Elena A. Kouzminova ◽

Andrei Kuzminov

Keyword(s):

Dna Replication ◽

Excision Repair ◽

Chromosomal Dna ◽

E Coli ◽

Okazaki Fragments ◽

Leading Strand ◽

Replication Intermediates ◽

Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

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

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2463-2474.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2463-2474 ◽

Cited By ~ 56

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.

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Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607552113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3639-E3648 ◽

Cited By ~ 14

Author(s):

Deepak Bastia ◽

Pankaj Srivastava ◽

Shamsu Zaman ◽

Malay Choudhury ◽

Bidyut K. Mohanty ◽

...

Keyword(s):

Replication Fork ◽

General Mechanism ◽

Nonhistone Protein ◽

In Vivo Experiments ◽

Vitro Binding ◽

Fork Protection Complex ◽

Replication Fork Arrest ◽

Replication Factors

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.923 ◽

1994 ◽

Vol 14 (2) ◽

pp. 923-933 ◽

Cited By ~ 180

Author(s):

M Foiani ◽

F Marini ◽

D Gamba ◽

G Lucchini ◽

P Plevani

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Cell Viability ◽

Dna Polymerase ◽

Chromosomal Dna ◽

Dna Polymerase Alpha ◽

B Subunit ◽

Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

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Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication

Molecular and Cellular Biology ◽

10.1128/mcb.01060-06 ◽

2006 ◽

Vol 26 (18) ◽

pp. 6993-7004 ◽

Cited By ~ 104

Author(s):

Christo P. Christov ◽

Timothy J. Gardiner ◽

Dávid Szüts ◽

Torsten Krude

Keyword(s):

Dna Replication ◽

Functional Characterization ◽

Noncoding Rnas ◽

Biological Processes ◽

Replication Forks ◽

Chromosomal Dna ◽

Free System ◽

Specific Degradation

ABSTRACT Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G1-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G1-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

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