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1. The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure

   https://doi.org/10.7554/eLife.61797  

   Chou, Hsiang-Chen ; Bhalla, Kuhulika ; Demerdesh, Osama EL ; Klingbeil, Olaf ; Hanington, Kaarina ; Aganezov, Sergey ; Andrews, Peter ; Alsudani, Habeeb ; Chang, Kenneth ; Vakoc, Christopher R ; et al ( February 2021 , eLife)

   null (Ed.)

   The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization. 

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2. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

   https://doi.org/10.7554/eLife.05795  

   Chang, FuJung ; Riera, Alberto ; Evrin, Cecile ; Sun, Jingchuan ; Li, Huilin ; Speck, Christian ; Weinreich, Michael ( August 2015 , eLife)

   To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

    

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3. Telomere-to-telomere human DNA replication timing profiles

   https://doi.org/10.1038/s41598-022-13638-8  

   Massey, Dashiell J. ; Koren, Amnon ( June 2022 , Scientific Reports)

   The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.

    

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4. Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint

   https://doi.org/10.1007/s00294-022-01228-0  

   Gasparayan, Hovik ; Caridi, Chris ; Julius, Jeff ; Feng, Wenyi ; Bachant, Jeff ; Nugent, Constance I. ( February 2022 , Current Genetics)

   Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13–Stn1–Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss ofRAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of functionstn1mutations compensaterad53S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth,stn1mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation atORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

    

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5. Image‐Based Elastography of Heterochromatin and Euchromatin Domains in the Deforming Cell Nucleus

   https://doi.org/10.1002/smll.202006109  

   Ghosh, Soham ; Cuevas, Victor Crespo ; Seelbinder, Benjamin ; Neu, Corey P. ( January 2021 , Small)

   Chromatin of the eukaryotic cell nucleus comprises microscopically dense heterochromatin and loose euchromatin domains, each with distinct transcriptional ability and roles in cellular mechanotransduction. While recent methods are developed to characterize the mechanics of nucleus, measurement of intranuclear mechanics remains largely unknown. Here, the development of “nuclear elastography,” which combines microscopic imaging and computational modeling to quantify the relative elasticity of the heterochromatin and euchromatin domains, is described. Using contracting murine embryonic cardiomyocytes, nuclear elastography reveals that the heterochromatin is almost four times stiffer than the euchromatin at peak deformation. The relative elasticity between the two domains changes rapidly during the active deformation of the cardiomyocyte in the normal physiological condition but progresses more slowly in cells cultured in a mechanically stiff environment, although the relative stiffness at peak deformation does not change. Further, it is found that the disruption of the Klarsicht, ANC‐1, Syne Homology domain of the Linker of Nucleoskeleton and Cytoskeleton complex compromises the intranuclear elasticity distribution resulting in elastically similar heterochromatin and euchromatin. These results provide insight into the elastography dynamics of heterochromatin and euchromatin domains and provide a noninvasive framework to further investigate the mechanobiological function of subcellular and subnuclear domains limited only by the spatiotemporal resolution of the acquired images.

    

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