pcdna 3 1 flag pol ii wt plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death

doi: 10.1101/2025.11.26.690880

Figure Lengend Snippet: Variants display higher affinity for CPD damaged DNA compared to WT Pol θ. WT POLQ and L2538R, E2406K, and T2161I variants were titrated from 0-1800 nM against 10 nM undamaged (circles) or damaged (triangles) dsDNA. Bound and unbound products were separated on a 6% non-denaturing gel and quantified using ImageQuant software. K D(DNA) was calculated using (Methods).

Article Snippet: WT POLQ rescue plasmids contained full-length WT POLQ obtained from Addgene (Addgene plasmid # 73132; http://n2t.net/addgene:73132 ; RRID:Addgene_73132, [ ]).

Techniques: Software

Journal: bioRxiv

Article Title: POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death

doi: 10.1101/2025.11.26.690880

Figure Lengend Snippet:

Article Snippet: WT POLQ rescue plasmids contained full-length WT POLQ obtained from Addgene (Addgene plasmid # 73132; http://n2t.net/addgene:73132 ; RRID:Addgene_73132, [ ]).

Techniques:

Journal: bioRxiv

Article Title: POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death

doi: 10.1101/2025.11.26.690880

Figure Lengend Snippet: WT Pol θ and cancer variants bypass and extend past CPD damaged DNA. A representative denaturing gel demonstrates primer extension of CPD dsDNA. Under single-turnover conditions 750 nM of WT POLQ and L2538R, E2406K, and T2161I variants were preincubated with 50 nM of CPD damaged dsDNA substrate and then combined with 10 mM MgCl 2 and either no dNTPs or 50 µM of All dNTPs, dATP, dCTP, dGTP, or dTTP for 5 minutes at 37°C. DNA extension products were separated on a denaturing gel and visualized on a Typhoon scanner. Each n+1 band represents extension past the CPD damage with either correct (dATP) or incorrect incorporation (dCTP, dGTP, or dTTP). Each additional band represents another extension with a maximum product of n+12.

Article Snippet: WT POLQ rescue plasmids contained full-length WT POLQ obtained from Addgene (Addgene plasmid # 73132; http://n2t.net/addgene:73132 ; RRID:Addgene_73132, [ ]).

Techniques:

Journal: bioRxiv

Article Title: POLQ variants with aberrant DNA polymerase activity protect against UV-induced cell death

doi: 10.1101/2025.11.26.690880

Figure Lengend Snippet: WT POLQ and variants E2406K, and T2161I reduce the number of double strand brakes in UV treated cells. MCF7 cells melanocytes were co-transfected with either a scrambled control siRNA oligo or an siRNA oligo to POLQ and either a siRNA resistant WT, L2538R, E2406K, or T2161I POLQ rescue plasmid. Cells were allowed to grow for 48-72 hrs. and then treated with either 5J ultra-violet radiation or not and allowed to recover for 24 hrs. Neutral comet assays were performed using Comet slides (n ≥ 50 cells) and analyzed using CometScore software. Error bars represent standard deviation, and significance was determined using One-way ANOVA (**** p < 0.0001).

Article Snippet: WT POLQ rescue plasmids contained full-length WT POLQ obtained from Addgene (Addgene plasmid # 73132; http://n2t.net/addgene:73132 ; RRID:Addgene_73132, [ ]).

Techniques: Transfection, Control, Plasmid Preparation, Software, Standard Deviation

Journal: medRxiv

Article Title: A germline heterozygous POLQ nonsense mutation causes hereditary colorectal cancer

doi: 10.1101/2024.01.13.23299913

Figure Lengend Snippet: a , POLQ mRNA level of POLQ +/R1953X cells increased early in response to DNA damage. b , POLQ expression level increased in POLQ +/R1953X HCT116 cell lines 2h after 6 Gy IR induction compared with POLQ +/+ cells. P -value was determined by two-way analysis of variance. c-d , Flow cytometric analysis of total-NHEJ, TMEJ and HR efficiency showed that POLQ R1953X mutation promoted TMEJ activity in HCT116 ( c ) and 293T ( d ) cells. e , Increased POLQ protein level and decreased DNA damage in POLQ +/R1953X HCT116 cells and 293T cells (f) treated with ultraviolet (UV) or ETOPOSIDE (ETOP), which is time-dependent. VINCILIN, TUBULIN and GADPH were used as the loading control. g-h , Reduced ETOP and UV induced DNA damages in POLQ +/R1953X 293T cells. DNA double-strand breaks measured by γ-H2AX level. Scale bars, 20µm. Data are shown as mean ± SD from at least three independent biological replicates. P -value was determined by two-way ANOVA. * P < 0.05; ** P < 0.01; *** P <0.001; **** P < 0.0001 and ns, not significant. i , Higher somatic mutation burden in cancer tissues derived from POLQ R1953X carriers, with frequently mutated oncogenes shown in ( j ).

Article Snippet: Myc and Flag-tagged POLQ expression plasmids were purchased from Addgene (#73132).

Techniques: Expressing, Mutagenesis, Activity Assay, Derivative Assay