Fluorescent indicators for simultaneous reporting of all four cell cycle phases

Bajar, Bryce T.; Lam, Amy; Badiee, Ryan K; Oh, Youjin; Chu, Jun; Zhou, Xin; Kim, Namdoo; Kim, Benjamin B.; Chung, Mingyu; Yablonovitch, Arielle; Cruz, Barney F; Kulalert, Kanokwan; Tao, Jacqueline J; Meyer, Tobias; Su, Xiao‐Dong; Lin, Michael Z.

doi:10.1038/nmeth.4045

Cited by 177 publications

(155 citation statements)

References 30 publications

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“…Furthermore, the PCNA-based approach also provides an accurate methodology crucial for locating cell cycle transitions and commitment points (Barr et al , 2016, 2017; Wilson et al , 2016). This method provides an advantage over the widely-used fluorescence ubiquitin cell cycle indicator (FUCCI) system (Dowling et al , 2014; Sandler et al , 2015; Wilson et al , 2016) and its newer version (FUCCI4) (Bajar et al , 2016). Whereas the identification of G1/S transition remained subjective and sensitive to thresholding in FUCCI system and is not capable of sharply demarcating the G1/S transition, the PCNA-based method provides a finer resolution demarking the boundaries at the G1/S and S/G2 borders (Wilson et al , 2016).…”

Section: Discussionmentioning

confidence: 99%

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle

et al. 2017

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Although molecular mechanisms that prompt cell cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S-phase progression. In sum, we show that a cell’s response to DNA damage depends on its exact cell cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none.

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Section: Discussionmentioning

confidence: 99%

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle

et al. 2017

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“…Primers used for molecular cloning are listed in the Appendix. To produce a cell cycle-regulatable RNase H1, a fragment of human geminin corresponding to amino acids 1-110 was PCR-amplified from pLL3.7m-Clover-Geminin(1-110)-IRES-mKO2-Cdt(30-120) from the Fucci4 system (Bajar et al, 2016) and cloned in frame to the C-terminus of chicken RNase H1 DMLS-YFP. cDNAs for PrimPol RPA binding mutants (ΔRBM-A and ΔRBM-B) were PCR-amplified from previously described vectors (Guilliam et al, 2017) with primers listed in the Appendix.…”

Section: Molecular Cloning and Transgene Constructsmentioning

confidence: 99%

“…Similarly, chicken RNase H1 (lacking the mitochondrial localisation sequence) was PCR-amplified from DT40 cDNA and cloned in frame with YFP on the C-terminus. To produce a cell cycle-regulatable RNase H1, a fragment of human geminin corresponding to amino acids 1-110 was PCR-amplified from pLL3.7m-Clover-Geminin(1-110)-IRES-mKO2-Cdt(30-120) from the Fucci4 system (Bajar et al, 2016) and cloned in frame to the C-terminus of chicken RNase H1 DMLS-YFP. The hybrid binding domain was amplified from human cDNA using primers previously published (Bhatia et al, 2014) and fused to C-terminal mCherry via flexible GSGSG linker.…”

Section: Molecular Cloning and Transgene Constructsmentioning

confidence: 99%

R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

Šviković

Crisp

Tan-Wong³

et al. 2018

The EMBO Journal

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During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R‐loops, three‐stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single‐stranded DNA, are important drivers of damage arising from such conflicts. How R‐loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R‐loop formation drives a short purine‐rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase‐polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R‐loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R‐loop formation in genes harbouring secondary structure‐forming sequences, exemplified by G quadruplex and H‐DNA motifs, across the genome in both avian and human cells. Thus, R‐loops promote the creation of replication blocks at susceptible structure‐forming sequences, while PrimPol‐dependent repriming limits the extent of unscheduled R‐loop formation at these sequences, mitigating their impact on replication.

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“…To visualize these proteins in host cells upon infection we generated Salmonella strains expressing the mRuby3 26 FP under control of the constitutive rpsM promoter, and harboring an isogenic deletion of the target effector protein. These strains were complemented by plasmid-based expression of a GFP11 tagged version of the effector.…”

Section: Resultsmentioning

confidence: 99%

Optimized Fluorescence Complementation Platform for Visualizing Salmonella Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types

et al. 2017

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The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG and SlrP, that have never before been visualized over time during infection of live cells. Using this platform, we developed a methodology for visualizing effector proteins in primary macrophage cells for the first time and reveal distinct differences in effector defined intracellular niche between primary macrophage and commonly used HeLa and RAW cell lines.

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Fluorescent indicators for simultaneous reporting of all four cell cycle phases

Cited by 177 publications

References 30 publications

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle

R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

Optimized Fluorescence Complementation Platform for Visualizing Salmonella Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types

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