New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.

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Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0426 ◽

2007 ◽

Vol 18 (1) ◽

pp. 129-141 ◽

Cited By ~ 53

Author(s):

Yasunari Takami ◽

Tatsuya Ono ◽

Tatsuo Fukagawa ◽

Kei-ichi Shibahara ◽

Tatsuo Nakayama

Keyword(s):

Dna Replication ◽

Essential Role ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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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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Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

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.

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Prokaryotic DNA replication mechanisms

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1987.0068 ◽

1987 ◽

Vol 317 (1187) ◽

pp. 395-420 ◽

Cited By ~ 57

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.

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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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Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.118.301853 ◽

2019 ◽

Vol 211 (3) ◽

pp. 877-892 ◽

Cited By ~ 7

Author(s):

Laura L. McCullough ◽

Trang H. Pham ◽

Timothy J. Parnell ◽

Zaily Connell ◽

Mahesh B. Chandrasekharan ◽

...

Keyword(s):

Nucleosome Occupancy ◽

Histone H3 ◽

Antisense Transcription ◽

Histone Chaperone ◽

Transcript Levels ◽

Chromatin Architecture ◽

Transcription Profiles ◽

Chromatin Integrity

FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone–DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity in vitro, and chromatin integrity and transcript output in vivo. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels. The results are consistent with emerging models that emphasize FACT’s importance in establishing global chromatin architecture prior to transcription, promoting transitions among different states as transcription profiles change, and restoring chromatin integrity after it is disturbed. Optimal FACT activity required the availability of both modified and unmodified states of H3-K56. Perturbing this balance was especially detrimental for maintaining repression of genes with high nucleosome occupancy over their promoters and for blocking antisense transcription at the +1 nucleosome. The results reveal a complex collaboration between H3-K56 modification status and multiple FACT functions, and support roles for nucleosome reorganization by FACT before, during, and after transcription.

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FACT activity and histone H3-K56 acetylation promote optimal establishment of chromatin architecture independent of ongoing transcription inSaccharomyces cerevisiae

10.1101/383984 ◽

2018 ◽

Author(s):

Laura McCullough ◽

Trang H. Pham ◽

Timothy J. Parnell ◽

Mahesh B. Chandrasekharan ◽

David J. Stillman ◽

...

Keyword(s):

Transcription Initiation ◽

Nucleosome Occupancy ◽

Histone H3 ◽

Antisense Transcription ◽

Transcript Levels ◽

Chromatin Architecture ◽

Nucleosome Structure ◽

Chromatin Integrity

AbstractFACT is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone:DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome structure, chromatin integrity, and transcription output. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACTin vitroandin vivoas expected, but H3-K56 and FACT mutations caused surprisingly similar changes in transcription of individual genes. Notably, neither the changes in transcript levels nor the effects on nucleosome occupancy resulting from mutations conformed to the model that FACT is needed to overcome nucleosomal barriers during transcription initiation or elongation. Instead, the results suggest that both FACT and H3-K56ac are involved in establishing chromatin architecture prior to transcription and restoring it afterwards. They contribute to a process that optimizes transcription frequency, especially at conditionally expressed genes, and restores chromatin integrity after transcription, especially at the +1 nucleosome to block antisense transcription, but FACT appears to be less involved than expected in directly promoting transcription.

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Two Fundamentally Distinct PCNA Interaction Peptides Contribute to Chromatin Assembly Factor 1 Function

Molecular and Cellular Biology ◽

10.1128/mcb.01051-09 ◽

2009 ◽

Vol 29 (24) ◽

pp. 6353-6365 ◽

Cited By ~ 44

Author(s):

Tom Rolef Ben-Shahar ◽

Araceli G. Castillo ◽

Michael J. Osborne ◽

Katherine L. B. Borden ◽

Jack Kornblatt ◽

...

Keyword(s):

Dna Replication ◽

Large Subunit ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Replication Forks ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

ABSTRACT Chromatin assembly factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.

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Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

10.1101/2021.02.25.432933 ◽

2021 ◽

Author(s):

Katie H. Jameson ◽

Christian J. Rudolph ◽

Michelle Hawkins

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Processing Enzymes ◽

Replication Fork Progression ◽

Evolutionary Advantage ◽

Domains Of Life ◽

Dna Template ◽

Template Dna

ABSTRACTThe complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the ‘replication fork trap’ region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

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The INO80 remodeller in transcription, replication and repair

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0290 ◽

2017 ◽

Vol 372 (1731) ◽

pp. 20160290 ◽

Cited By ~ 32

Author(s):

Jérôme Poli ◽

Susan M. Gasser ◽

Manolis Papamichos-Chronakis

Keyword(s):

Dna Repair ◽

Rna Polymerase Ii ◽

Replication Fork ◽

Oxidative Dna Damage ◽

Structural Features ◽

Atpase Subunit ◽

Replication Fork Progression ◽

Chromatin Template

The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. In vitro studies showed that INO80 promotes replication fork progression on a chromatin template, while in vivo it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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