Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: Recruitment of KAP1 to EA-D at replication forks requires transcription. ( A ) BL cells were exposed to NaB for 30h with α-amanitin added for the last 6h. Cells were then harvested for iPOND followed by immunoblotting. YC, yes click group, processed with biotin azide; NC, no click control group, processed without biotin azide. ( B, C, E, F ) EBV in BL cells was reactivated using NaB for 24 h while blocking transcription using α-amanitin for the last 6 h. Cells were harvested for immunoprecipitation with anti-EA-D antibody (versus IgG as control; (B), immunoblotting (C), quantitation of viral genomes by qPCR (E) and quantitation of immediate early and early gene transcripts via RT-qPCR (F). In panel D , BL cells were exposed to NaB for 30 h with α-amanitin added for the last 6h. DNA was precipitated using antibody to γH2AX and exposed to MboI to digest newly replicated non-methylated DNA. Primer pairs spanning MboI sites were used to amplify indicated loci on episomal genomes using qPCR. Efficiency of MboI digestion was assayed by qPCR using a pair of primers spanning an MboI site on the BALF5 gene (middle and right-hand side panels). Error bars, SEM of three technical replicates. Experiments were performed twice.

Article Snippet: si KAP1 #1 , Santa Cruz , sc-38550.

Techniques: Western Blot, Control, Blocking Assay, Immunoprecipitation, Quantitation Assay, Quantitative RT-PCR, Methylation

Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: KAP1 conjugates SUMO2 to polymerase processivity factor EA-D at K135 during DNA replication. ( A, B ) BL cells (A) and LCL (B) were treated with NaB with or without PAA for 36 h and analyzed using iPOND for active forks (EdU) and mature newly replicated DNA (using thymidine) and immunoblotted with indicated antibodies. Click group was processed with biotin azide. ( C ) BL cells exposed to NaB for 36 h were then labeled with BrdU for 2 h. Harvested KAP1 hi and KAP1 −/lo cells (left two panels) were assayed for BrdU uptake (right two panels) using flow cytometry. ( D ) HEK-293T cells were transfected with FLAG- BMRF1 (EA-D), FLAG-KAP1, FLAG-SENP1 and HA-SUMO2. After 48 h, lysates were immunoprecipitated with anti-EA-D antibody prior to immunoblot analyses. ( E ) 293-EBV cells were co-transfected with siRNA targeting KAP1 alongside FLAG- BZLF1 and FLAG- BRLF1 to reactivate EBV. After 36 h, lysates were immunoprecipitated with anti-SUMO2 antibody or control IgG and then immunoblotted. ( F ) HEK-293T cells were transfected with wild type FLAG- BMRF1 (WT) or mutants (K135R, K212R) together with FLAG-KAP1 and HA-SUMO2 for 48 h. Lysates were immunoprecipitated with anti-EA-D antibody or control IgG and immunoblotted as indicated. ( G, H ) BL cells were treated with NaB for 24 h and lysates were immunoprecipitated with anti-EA-D antibody versus control IgG (G) or anti-SUMO2 antibody versus control IgG (H), and then immunoblotted as indicated. ( I–K ) To assess proximity by proximity ligation assay (PLA), LCL were exposed to NaB without (I, J) or with (K) the addition of PAA for 36 h; lytic cells were marked using an anti-ZEBRA antibody. ( L ) LCL were exposed to NaB with or without the addition of PAA for 36 h prior to immunoprecipitation using an anti-KAP1 antibody or control IgG followed by immunoblotting. ( M ) BL cells were exposed to NaB for different lengths of time prior to immunoprecipitation with anti-EA-D antibody versus control IgG and then immunoblotted. Experiments were performed three times.

Article Snippet: si KAP1 #1 , Santa Cruz , sc-38550.

Techniques: Labeling, Flow Cytometry, Transfection, Immunoprecipitation, Western Blot, Control, Proximity Ligation Assay

Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: KAP1 mediated SUMOylation of EA-D stabilizes viral DNA replication forks. ( A ) EA-D/ BMRF1 -deleted EBV bacmid-carrying 293-EBV (B072-1) cells were transfected with wild type FLAG- BMRF1 (WT) or mutants (K135R, K212R) together with FLAG- BZLF1 and FLAG- BRLF1 to reactivate EBV. After 36 h, viral genomes were quantified via qPCR. (B–E) 293-EBV cells were transfected with si KAP1 (versus siCtrl) and FLAG- BZLF1 and FLAG- BRLF1 for 36 h and extracted DNA subjected to DNA fiber fluorography ( B–D ) or lysates immunoblotted with indicated antibodies ( E ). Representative images from DNA fiber analysis (B), fiber counts (C) and cumulative data including median value of track/fiber length from at least 150 tracks per experimental condition (D) are shown. * P < 0.05, *** P < 0.001. ( F ) BMRF1 -KO 293-EBV cells were transfected with wild type FLAG- BMRF1 (WT) or mutants (K135R, K212R) together with FLAG- BZLF1 and FLAG- BRLF1 plasmids for 36 h prior to performing immunoblotting. Experiments were performed at least twice. Error bars in (A), SEM of three technical replicates.

Article Snippet: si KAP1 #1 , Santa Cruz , sc-38550.

Techniques: Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: The PI3KK ATM recruits and phosphorylates KAP1 at the RPOII-RECQ5 complex. ( A, B ) BL cells were treated with NaB with or without the ATM inhibitor KU55933 for 24 h. Cells were harvested for immunoblotting (A) or immunoprecipitation with anti-EA-D antibody or control IgG (B). ( C, D ) BL cells were transfected with si ATM or scrambled siRNA (siCtrl) for 20 h followed by exposure to NaB for 24 h. Lysates were immunoprecipitated with anti-RECQ5 antibody (C) or anti-KAP1 antibody (D) and then immunoblotted using indicated antibodies. Experiments were performed twice.

Article Snippet: si KAP1 #1 , Santa Cruz , sc-38550.

Techniques: Western Blot, Immunoprecipitation, Control, Transfection

Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: si KAP1 #1 , Santa Cruz , sc-38550.

Techniques: Purification, Gel Extraction, DNA Extraction, RNA Extraction, Plasmid Preparation, SYBR Green Assay, Electroporation, In Vitro, Transfection, Recombinant