TIF1 Represses rDNA Replication Initiation, but Promotes Normal S Phase Progression and Chromosome Transmission in Tetrahymena

The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication. Despite this, replication of the rDNA minichromosome initiates precociously. Because rDNA copy number is unaffected in the polyploid macronucleus, mechanisms that prevent reinitiation appear intact. Although mutants exit macronuclear S with a wild-type DNA content, division of the amitotic macronucleus is both delayed and abnormal. Nuclear defects are also observed in the diploid mitotic micronucleus, as TIF1 mutants lose a significant fraction of their micronuclear DNA. Hence, TIF1 is required for the propagation and subsequent transmission of germline chromosomes. The broad phenotypes associated with a TIF1-deficiency suggest that this origin binding protein is required globally for the proper execution and/or monitoring of key chromosomal events during S phase and possibly at later stages of the cell cycle. We propose that micro- and macronuclear defects result from exiting the respective nuclear S phases with physically compromised chromosomes.

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The limiting DNA replication initiation factors Sld2 and Sld3 influence origin efficiency independent of origin firing time

10.1101/384644 ◽

2018 ◽

Author(s):

Kelsey L. Lynch ◽

Elizabeth X. Kwan ◽

Gina M. Alvino ◽

Bonita J. Brewer ◽

M.K. Raghuraman

Keyword(s):

Rdna Locus ◽

S Phase ◽

Replication Initiation ◽

Yeast Genome ◽

Phase Duration ◽

Origin Firing ◽

Dna Replication Initiation ◽

Initiation Factors ◽

Origin Activation ◽

Time Of Origin

AbstractChromosome replication in Saccharomyces cerevisiae is initiated from roughly 300 origins that are regulated both by DNA sequence and by the limited abundance of four trans-acting initiation proteins (Sld2, Sld3, Dpb11 and Dbf4, collectively called “SSDD”). We set out to determine how the association of Sld2 or Sld3 at origins contributes to time of origin activation and/or origin efficiency using auxin-induced protein degradation to further decrease their abundance. Depleting cells of either factor slows growth rate, increases S-phase duration, and causes viability defects, without activating the S phase checkpoint. Chr XII is uniquely unstable with breakage occurring specifically within the rDNA locus. The efficiency of the rDNA origin is decreased while the onset of replication initiation is unchanged. We found that origin efficiency is reduced uniformly across the unique portions of the yeast genome. We conclude that the abundance of Sld2 and Sld3 contribute primarily to origin efficiency.

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Precocious S-Phase Entry in Budding Yeast Prolongs Replicative State and Increases Dependence Upon Rad53 for Viability

Genetics ◽

10.1093/genetics/160.1.123 ◽

2002 ◽

Vol 160 (1) ◽

pp. 123-136

Author(s):

Julia M Sidorova ◽

Linda L Breeden

Keyword(s):

Kinase Activity ◽

Growth Conditions ◽

S Phase ◽

Wild Type ◽

Origin Firing ◽

Phase Progression ◽

Rad53 Kinase ◽

Replication Intermediates ◽

Phase Entry ◽

In Cells

Abstract Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.

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CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

Biochemical Journal ◽

10.1042/bj3400135 ◽

1999 ◽

Vol 340 (1) ◽

pp. 135-141 ◽

Cited By ~ 23

Author(s):

Parisa DANAIE ◽

Michael ALTMANN ◽

Michael N. HALL ◽

Hans TRACHSEL ◽

Stephen B. HELLIWELL

Keyword(s):

Cell Cycle ◽

S Phase ◽

Translation Initiation Factor ◽

Yeast Cells ◽

Initiation Factor ◽

G1 Phase ◽

Mrna Levels ◽

Wild Type ◽

Phase Progression ◽

Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

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Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0020 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4374-4382 ◽

Cited By ~ 13

Author(s):

Ling Yin ◽

Alexandra Monica Locovei ◽

Gennaro D'Urso

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

S Phase ◽

Replication Initiation ◽

Dna Damage Checkpoint ◽

Origin Firing ◽

Dna Replication Initiation ◽

Fork Stalling ◽

S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

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Activation of mammalian Chk1 during DNA replication arrest

The Journal of Cell Biology ◽

10.1083/jcb.200104099 ◽

2001 ◽

Vol 154 (5) ◽

pp. 913-924 ◽

Cited By ~ 220

Author(s):

Carmen Feijoo ◽

Clare Hall-Jackson ◽

Rong Wu ◽

David Jenkins ◽

Jane Leitch ◽

...

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

S Phase ◽

Global Pattern ◽

Origin Firing ◽

Replication Arrest ◽

Molecule Inhibitor ◽

Replication Block ◽

Phase Progression ◽

S Phase Checkpoint

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

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Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2809-2817.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2809-2817 ◽

Cited By ~ 115

Author(s):

Hiroshi Masumoto ◽

Akio Sugino ◽

Hiroyuki Araki

Keyword(s):

Dna Polymerases ◽

S Phase ◽

Wild Type ◽

Chromosomal Dna ◽

Origin Firing ◽

Autonomously Replicating Sequence ◽

Sequence Region ◽

Late Origin ◽

Checkpoint Genes ◽

Primase Complex

ABSTRACT Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase ɛ (Dpb11-Polɛ complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polɛ associated with these fragments. Association of Dpb11 and Polɛ with these fragments was mutually dependent, suggesting that the Dpb11-Polɛ complex associates with the ARS. Moreover, Dpb11 was required for the association of Polα-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polɛ complex with the ARS fragments is required for the association of the Polα-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polɛ in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases α and ɛ and the ARS.

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Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p

Genes & Development ◽

10.1101/gad.14.4.493 ◽

2000 ◽

Vol 14 (4) ◽

pp. 493-503 ◽

Cited By ~ 2

Author(s):

Rita S. Cha ◽

Beth M. Weiner ◽

Scott Keeney ◽

Job Dekker ◽

N. Kleckner

Keyword(s):

Dna Replication ◽

Phase Modulation ◽

Cell Types ◽

Strand Break ◽

S Phase ◽

Wild Type ◽

Homolog Pairing ◽

Defective Mutant ◽

Phase Progression ◽

Phase Length

Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion of the SPO11 gene decreases the length of S phase by ∼25%. Rec8p is a key coordinator of meiotic interhomolog and intersister interactions. Deletion of the REC8 gene increases S-phase length, by ∼10% in wild-type and ∼30% in aspo11Δ background. Thus, the progression of DNA replication is modulated by interchromosomal interaction proteins. Thespo11–Y135F DSB (double strand break) catalysis-defective mutant is normal for S-phase modulation and DSB-independent homolog pairing but is defective for later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and later functions of Spo11 are defined. We propose that meiotic S-phase progression is linked directly to development of specific chromosomal features required for meiotic interhomolog interactions and that this feedback process is built upon a more fundamental mechanism, common to all cell types, by which S-phase progression is coupled to development of nascent intersister connections and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11 in progression through other stages of meiosis are also revealed.

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Transcription drives DNA replication initiation and termination in human cells

10.1101/324079 ◽

2018 ◽

Author(s):

Yu-Hung Chen ◽

Sarah Keegan ◽

Malik Kahli ◽

Peter Tonzi ◽

David Fenyö ◽

...

Keyword(s):

Dna Replication ◽

Rna Polymerase Ii ◽

Replication Fork ◽

Human Cells ◽

Replication Initiation ◽

Origin Firing ◽

Dna Replication Initiation ◽

Origin Activation ◽

Dna Replication Origins ◽

The Impact

ABSTRACTThe locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

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Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

10.1101/2020.09.30.320325 ◽

2020 ◽

Author(s):

Mark C. Johnson ◽

Geylani Can ◽

Miguel Santos ◽

Diana Alexander ◽

Philip Zegerman

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

S Phase ◽

Dna Lesions ◽

Replication Initiation ◽

Origin Firing ◽

Initiation Factors ◽

Dependent Inhibition ◽

Rad53 Kinase ◽

S Phase Checkpoint

AbstractAcross eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

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The Mre11 Complex Mediates the S-Phase Checkpoint through an Interaction with Replication Protein A

Molecular and Cellular Biology ◽

10.1128/mcb.00532-07 ◽

2007 ◽

Vol 27 (17) ◽

pp. 6053-6067 ◽

Cited By ~ 51

Author(s):

Erin Olson ◽

Christian J. Nievera ◽

Enbo Liu ◽

Alan Yueh-Luen Lee ◽

Longchuan Chen ◽

...

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Protein A ◽

Replication Protein A ◽

S Phase ◽

Replication Initiation ◽

Replication Protein ◽

Direct Role ◽

Origin Firing ◽

S Phase Checkpoint

ABSTRACT The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

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