Journal: Nature Communications

Article Title: CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion

doi: 10.1038/s41467-021-22927-1

Figure Lengend Snippet: a Scheme depicting a generic CopyCatcher element. Light blue boxes: exons of a targeted gene on the donor chromosome; dark blue boxes: exons on the receiver chromosome; black lines: genomic DNA; red box: splice acceptor site (SA); yellow box: T2A self-cleavage peptide; light red arrow: DsRed reporter; dark purple arrow: gRNA; light blue arrow: selection marker mCerulean ; red hourglass marks: point mutations at or near initiator ATG codons of endogenous genes; short black line: Cas9/gRNA cleavage site; light blue circle and dark lines: Cas9/gRNA complex. Scissors in ( a ) denote insertion of cargo cassettes into DSBs on homologous chromosomes including SA, T2A, gRNA, and selection marker mCerulean . b – d Distinct outcomes of three DSB repair mechanisms followed by Cas9/gRNA cleavage of the homologous “receiver” chromosome. Slashes on the second exons in ( b )–( d ) indicate loss of function on the marked chromosomes. Labels indicate resulting phenotypes. e, f Mosaic clones of somatic gene conversion (SGC) in two CopyCatcher lines. Photographs show the phenotypes and fluorescence patterns of two CopyCatcher elements inserted into the intron of white and ple loci in flies without (( e ) w [3XP3-CC] , ( f ) ple [CC] ) or with (( e ) w [ATG-,3XP3-CC] , ( f ) ple [ATG-,CC] ) associated ATG – point mutations, or F1 mosaics resulting from Cas9-mediated copying (( e ) w [ATG-,3XP3-CC] / w +; Cas9/+, ( f ) ple [ATG-,CC] /Cas9). SGC clones generated by ple [ATG-,CC] are outlined with white dotted lines. Rightmost panels in ( e ) and ( f ) are higher magnification views of areas delineated by white boxes in the lower magnification views immediately to their left. At least five independent flies were imaged and observed in ( e ) and ( f ) with similar results. Scale bars stand for 150 pixels in ( e ) and ( f ).

Article Snippet: The SA-T2A sequence was amplified from pBS-KS-attB2-SA-T2A-3XGal80-Hsp70 series plasmids (Addgene 62951 and 62952) , gRNAs-expressing cassette targeting to the first intron of yellow , white , and ple were assembled following online published protocols (CRISPR fly Design - http://www.crisprflydesign.org/ ) and mCerulean selection marker was amplified from Addgene 27795 plasmid.

Techniques: Selection, Marker, Clone Assay, Fluorescence, Generated

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Pericentromeric cohesin is deprotected long before sister chromatids separate. During phosphorylation- and Wapl-dependent release of cohesin from chromosome arms, pericentromeric cohesin is protected by associated Sgo1-PP2A. Following the early mitotic degradation of Nek2a and cyclin A2, Sgo1-PP2A relocates to kinetochores. Yet, pericentromeric cohesion persists until securin and cyclin B1 are degraded and separase is activated. A requirement for Nek2a and cyclin A2 for prophase pathway signaling would explain why, in metaphase, Wapl can no longer release cohesin. ( B ) Nek2a and Cdk1/2-cyclin A2 are necessary for Wapl-dependent in vitro displacement of cohesin from chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, kinases, and inhibitors (BI2536, ZM-447439, and RO-3306), as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. ( C ) Wapl and the three kinases Nek2a, Cdk1/2-cyclin A2, and Aurora B-INCENP are sufficient to support the displacement of cohesin from isolated chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, Nek2a, Cdk1/2-cyclin A2, Cdk1-cyclin B1, aurora B, and/or Plk1, as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Purification, Sedimentation, Incubation, Recombinant, Western Blot, Isolation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Recombinant kinases are active as judged by the phosphorylation of model substrates. Plk1, aurora B-INCENP, Nek2a, Cdk1/2-cyclin A2, or Cdk1-cyclin B1 supplemented with their specific inhibitor or carrier solvent DMSO (−) were incubated with the corresponding model substrate in the presence of [γ 33 P]-ATP. Reactions were subjected to SDS-PAGE followed by autoradiography. BI BI2536, ZM ZM-447439, ST staurosporine, RO RO-3306, MBP myelin basic protein. ( B ) Recombinant Nek2a, Cdk1/2-cyclin A2, and Wapl are free of cyclin B1 and Plk1. The preparations of Nek2a, Cdk1/2-cyclin A2, Wapl, Cdk1-cyclin B1, and Plk1 used for the release assays were characterized by immunoblotting using the indicated antibodies. In vitro expressed (IVTT) cyclin B1 served as an additional control.

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Solvent, Incubation, SDS Page, Autoradiography, Western Blot, In Vitro, Control

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Experimental outline. DNA-mediated chromatin pull-down from G2-arrested HeLaK cells is followed by cohesin eviction experiments. ( B ) Cohesin is eluted from immobilized chromatin upon DNA-cleavage but not high salt treatment. Chromatin beads were treated with benzonase and/or high salt, and retained proteins were detected by immunoblotting. High-salt-washed chromatin (lane 6) was used for subsequent cohesin eviction experiments. ( C ) Wapl-dependent cohesin release in vitro requires Nek2a, Cdk1/2-cyclin A2, and Aurora B but not Cdk1-cyclin B1 and Plk1. Immobilized chromatin was combined with the indicated proteins. Following centrifugation, DNA-beads and supernatant were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Western Blot, In Vitro, Centrifugation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Nek2a and Cdk1/2-cyclin A2 target juxtaposed but distinct residues within the disordered, C-terminal domain of Pds5b. In vitro expressed Pds5b variants were incubated with the indicated kinases and inhibitors in the presence of [γ- 33 P]-ATP, subjected to SDS-PAGE, and analyzed by autoradiography and immunoblotting. Note that Pds5b is also phosphorylated by aurora B and, weakly, by Plk1. KD kinase-dead Nek2a-Lys37Met; IVTT coupled in vitro transcription-translation. ( B ) Graphical summary of Nek2a- and Cdk1/2-cyclin A2-targeted Pds5b residues and corresponding phosphorylation-resistant variants. ( C ) Ser1209 of Pds5b is phosphorylated by Nek2a but not Cdk1/2-cyclin A2. Following incubation of in vitro expressed Pds5b-WT or −3A with Nek2a or Cdk1/2-cyclin A2, samples were analyzed by immunoblotting using the indicated antibodies. Nek2a-Lys37Met (KD, kinase-dead) served as a negative control. ( D ) The spatiotemporal dynamics of Ser1209-phosphorylation correlate with the recruitment of Wapl to chromatin followed by cohesin release. HeLaK cells synchronously cycling through mitosis were subjected to time-resolved fractionation and immunoblotting. At the time of release from a RO-3306-mediated arrest, cultures were supplemented with a Nek2a inhibitor (NCL00017509) or carrier solvent (mock). .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Western Blot, Negative Control, Fractionation, Solvent

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Transduction, Sequencing, Protease Inhibitor, Membrane, Software