Abstract 5187: The membrane traffic regulator PACS-1 mediates genome stability at S-phase of the cell cycle.

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

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Precise detection of S phase onset reveals decoupled G1/S transition events

10.1101/300442 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gavin D. Grant ◽

Katarzyna M. Kedziora ◽

Juanita C. Limas ◽

Jeremy E. Purvis ◽

Jeanette Gowen Cook

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Cycle Progression ◽

S Phase ◽

Cycle Phase ◽

Reporter System ◽

Phase Boundaries ◽

Fluorescent Reporter ◽

Molecular Events ◽

Cell Cycle Phases

AbstractThe eukaryotic cell division cycle is the process by which cells duplicate their genomes and proliferate. Transitions between sequential cell cycle phases are tightly orchestrated to ensure precise and efficient cell cycle progression. Interrogating molecular events at these transitions is important for understanding normal and pathological cell proliferation and mechanisms that ensure genome stability. A popular fluorescent reporter system known as “FUCCI” has been widely adopted for identifying cell cycle phases. Using time-lapse fluorescence microscopy, we quantitatively analyzed the dynamics of the FUCCI reporters relative to the transitions into and out of S phase. Although the original reporters reflect the E3 ubiquitin ligase activities for which they were designed, SCFSkp2 and APCCdh1, their dynamics are significantly and variably offset from actual S phase boundaries. To precisely mark these transitions, we generated and thoroughly validated a new reporter containing a PCNA-interacting protein degron whose oscillations are directly coupled to the process of DNA replication itself. We combined this reporter with the geminin-based APCCdh1 reporter to create “PIP-FUCCI.” PIP degron reporter dynamics closely correlate with S phase transitions irrespective of reporter expression levels. Using PIP-FUCCI, we made the unexpected observation that the apparent timing of APCCdh1 inactivation frequently varies relative to the onset of S phase. We demonstrate that APCCdh1 inactivation is not a strict pre-requisite for S phase entry, though delayed APCCdh1 inactivation correlates with longer S phase. Our results illustrate the benefits of precise delineation of cell cycle phase boundaries for uncovering the sequences of molecular events at critical cell cycle transitions.

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p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability

Nature Communications ◽

10.1038/s41467-020-18838-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Longtao Wu ◽

Clayton D. Crawley ◽

Andrea Garofalo ◽

Jackie W. Nichols ◽

Paige-Ashley Campbell ◽

...

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Cycle Progression ◽

Human Cancer ◽

S Phase ◽

Post Translational Modification ◽

Chromosomal Breakage ◽

Cycle Progression ◽

Genome Wide ◽

Phase Progression

Abstract p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

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Transcriptional Regulation of thep53Tumor Suppressor Gene in S-Phase of the Cell-Cycle and the Cellular Response to DNA Damage

Biochemistry Research International ◽

10.1155/2012/808934 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 24

Author(s):

David Reisman ◽

Paula Takahashi ◽

Amanda Polson ◽

Kristy Boggs

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Transcriptional Regulation ◽

Cellular Response ◽

Genome Stability ◽

Suppressor Gene ◽

S Phase ◽

Dna Binding Factors ◽

Combined Action ◽

Cellular Stressors

Thep53tumor suppressor induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors and thus participates in maintaining genome stability. Numerous studies have demonstrated thatp53transcription is activated before or during early S-phase in cells progressing from G0/G1into S-phase through the combined action of two DNA-binding factors RBP-Jκand C/EBPβ-2. Here, we review evidence that this induction occurs to provide availablep53mRNA in order to prepare the cell for DNA damage in S-phase, this ensuring a rapid response to DNA damage before exiting this stage of the cell cycle.

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Biphasic Incorporation of Centromeric Histone CENP-A in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0504 ◽

2008 ◽

Vol 19 (2) ◽

pp. 682-690 ◽

Cited By ~ 75

Author(s):

Yuko Takayama ◽

Hiroshi Sato ◽

Shigeaki Saitoh ◽

Yuki Ogiyama ◽

Fumie Masuda ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Genome Stability ◽

Fluorescent Protein ◽

Gene Activation ◽

S Phase ◽

Dependent Manner ◽

Gata Factor ◽

Histone H3 Variant ◽

Green Fluorescent

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Δams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Δams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Δams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged.

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Efficiency and equity in origin licensing to ensure complete DNA replication

Biochemical Society Transactions ◽

10.1042/bst20210161 ◽

2021 ◽

Author(s):

Liu Mei ◽

Jeanette Gowen Cook

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Genome Stability ◽

S Phase ◽

Replication Initiation ◽

G1 Phase ◽

Dna Replication Initiation ◽

Origin Licensing ◽

Dna Replication Origins

The cell division cycle must be strictly regulated during both development and adult maintenance, and efficient and well-controlled DNA replication is a key event in the cell cycle. DNA replication origins are prepared in G1 phase of the cell cycle in a process known as origin licensing which is essential for DNA replication initiation in the subsequent S phase. Appropriate origin licensing includes: (1) Licensing enough origins at adequate origin licensing speed to complete licensing before G1 phase ends; (2) Licensing origins such that they are well-distributed on all chromosomes. Both aspects of licensing are critical for replication efficiency and accuracy. In this minireview, we will discuss recent advances in defining how origin licensing speed and distribution are critical to ensure DNA replication completion and genome stability.

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Fungal zygote survival and ploidy maintenance rely on cell cycle-dependent and independent mating blocks

10.1101/2020.07.26.222000 ◽

2020 ◽

Author(s):

Aleksandar Vještica ◽

Melvin Bérard ◽

Gaowen Liu ◽

Laura Merlini ◽

Pedro Junior Nkosi ◽

...

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Cycle Progression ◽

S Phase ◽

Cycle Progression ◽

Mating Behaviors ◽

Cycle Dependent ◽

Necessary And Sufficient ◽

Meiotic Cycle

AbstractTo ensure genome stability, sexually reproducing organisms require that mating brings together exactly two haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins post-fusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least two mechanisms where the zygotic fate imposed by Mei2 and the cell cycle re-entry triggered by Mei3 synergize to prevent zygotic mating.

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Genome duplication in Leishmania major relies on DNA replication outside S phase

10.1101/799429 ◽

2019 ◽

Author(s):

Jeziel D. Damasceno ◽

Catarina A. Marques ◽

Dario Beraldi ◽

Kathryn Crouch ◽

Craig Lapsley ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Genome Stability ◽

Leishmania Major ◽

Genome Duplication ◽

S Phase ◽

Replication Initiation ◽

Dna Replication Initiation ◽

Synthesis Activity ◽

Important Parasite

AbstractOnce every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase. Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex. Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite.

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Phosphorylation of the RB C-terminus regulates condensin II release from chromatin

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016511 ◽

2020 ◽

pp. jbc.RA120.016511

Author(s):

Seung J Kim ◽

James I MacDonald ◽

Frederick A. Dick

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Cycle Control ◽

Cell Receptor ◽

Receptor Activation ◽

Biologically Relevant ◽

C Terminus ◽

Independent Functions ◽

Phosphorylation Event ◽

Rb Phosphorylation

The retinoblastoma tumour suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate, and render RB inactive in late G1/S, promoting entry into S phase. Recently, mono-phosphorylated RB species were described to have distinct cell-cycle independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output. We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK, and RB S838/T841 phosphorylation. This signaling pathway disrupts RB and condensin II interaction with chromatin. Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.

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