rabbit anti polλ  (Bethyl)

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, Schematic of MMEJ. Following 5′−3′ DNA resection, Polθ promotes microhomology-mediated synapsis of 3′ ssDNA overhangs then performs strand extension. b, Schematic of pssDNA 344/343P. Red text, microhomology; P, phosphate (top). Nondenaturing gel showing MMEJ activity by the indicated Pols. MMEJ (%) is shown at the bottom; a, abortive end-joining. c, Nondenaturing gel showing MMEJ activity by the indicated Pols. d, Nondenaturing gel showing XmaI digestion of the MMEJ product formed by Polλ. e,f, Nondenaturing gels showing a time course of MMEJ by Polλ (e) and Polθ (f). Experiments in b−f were all repeated with three independent samples and all yielded similar results. g, Scatter plot showing the relative rates of MMEJ by Polθ and Polλ; n = 3 independent samples, ± s.d. Relative steady-state rates of DNA joined by MMEJ are indicated.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Activity Assay

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, Denaturing gels showing extension of the indicated primer-template by the indicated Pols using identical conditions. Polμ is known to require a downstream ssDNA strand for optimal activity primer extension activity along a short gap. 20 nM Pol concentrations were used. b, Schematic of DNA templates. Microhomology indicated as red text. c, Non-denaturing gel showing Polλ MMEJ as a positive control for its activity (left panel). Denaturing gels showing extension of the indicated templates in the presence of all 4 dNTPs by the indicated Pols (middle and right panel). 20 nM Pol concentrations were used. d, Schematic showing the respective activities of Polθ and Polλ on the indicated templates. although both enzymes perform MMEJ (top), only Polθ exhibits ssDNA extension due to its snap-back replication activity.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Activity Assay, Positive Control

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, Schematic showing pssDNA modifications. b, Schematic of pssDNA and ssDNA templates. Red text, microhomology; asterisk, radiolabel; P, 5′-phosphate. c, Nondenaturing gel showing MMEJ by Polθ and Polλ on the indicated templates with and without 5′-phosphate on the resected strand. MMEJ (%) indicated. Experiment was repeated with three independent samples and all yielded similar results. d, Bar plot showing MMEJ (%) by Polλ on the indicated templates. Data represent means; n = 3 independent samples, ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance from two-sample t-test between 344/343 and 344/343P, P = 0.0008. e,f, Nondenaturing gels showing MMEJ by Polλ (e) and Polθ (f) on the indicated templates. Experiment was repeated with three independent samples and all yielded similar results. g, Bar plot showing MMEJ (%) by Polλ and Polθ on the indicated templates. Data represent means; n = 3 independent samples, ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance from two-sample t-test between 362/343P and 602/343P for Polλ, P = 0.0005. h, Nondenaturing gel showing MMEJ activity by Polλ and Polθ on the indicated template. Bar plot showing MMEJ (%) by Polλ and Polθ on the indicated template (right). Data represent means; n = 3 independent samples, ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance from two-sample t-test between Polθ and Polλ, P = 0.0006. i, Nondenaturing gel showing MMEJ and snapback replication activities by Polλ and Polθ on the indicated template. j, Summary table comparing the respective MMEJ activities of Polλ and Polθ on different templates.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Activity Assay

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, Denaturing gel showing extension of a pssDNA substrate containing 2 bp of microhomology in the presence of dCTP. Polλ but not Polμ performs addition of dCMP on the indicated substrate. Microhomology indicated as red text. 20 nM Pol concentrations were used. b, Non-denaturing gel showing MMEJ activity by the indicated Pols on the indicated pssDNA containing 6 bp of microhomology (red text). Polλ performs MMEJ whereas Polβ does not. Reactions were performed in duplicate. 20 nM Pol concentrations were used.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Activity Assay

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, Top, schematic of MMEJ reporter containing 5′-streptavidin-biotin linkages. Middle, internal termini of left and right MMEJ reporter DNA constructs. Bottom, schematic of MMEJ reporter assay. b−f, Bar plots showing relative GFP frequencies following cotransfection of left and right MMEJ reporter DNA constructs, with immunoblots showing abundance of protein shown in c and e−g. b, GFP+ frequencies are shown relative to nontargeting siRNA (siControl = 1) in wildtype HEK293T cells; n = 3; P = 0.01. c, GFP+ frequencies relative to POLλ+/+ 293T cells (POLλ+/+ = 1). n = 3, P = 0.01. d, Same as in b in POLλ−/− 293T cells. n = 3, P = 0.03. e, GFP+ frequencies relative to nontargeting siRNA (siControl = 1). n = 3, P = 0.04. f, GFP+ frequencies relative to nontargeting siRNA (siControl = 1) in XRCC4−/− 293T cells. Data represent means. n = 2, P = 0.04. g, MMEJ GFP reporter assay. Schematic of GFP reporter assay (top). Bar plot of percentage of GFP cells following transient expression of I-SceI and cotransfection of either Polλ siRNA or Control siRNA. n = 2, P = 0.04. h, Bar plots showing percentage of colonies relative to control after siRNA transfection in DLD1 BRCA2−/− or DLD1 Parental cells (top), in MDA-MB-436 BRCA1 mut or MDA-MB-231 cells (bottom). Percentage of colonies are normalized to nontargeting siRNA (siControl = 100). n = 1. Colony images are on the right. In b and c−g, GFP+ frequencies are normalized to transfection efficiency. Data represent means. ‘n’ denotes number of independent experiments with triplicates for each condition, ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was measured from two-sample t-test and P values are indicated.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Construct, Reporter Assay, Cotransfection, Western Blot, Expressing, Control, Transfection

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a. RT qPCR analysis of Polθ expression. mRNA levels were corrected with internal control for Actin in siRNA-treated cells used in Fig. 3b, ​,dd as well as normalized to non-targeting siRNA (siControl = 1). Data represent mean. n = 1 experiment with triplicate for each condition ±SEM. b. gRNA sequence used to generate POLL−/− HEK293T cells via CRISPR-Cas9 engineering. Schematic representation of three isoforms of human Polλ with protein domains as well as location of gRNA sequence (red) is indicated. The genome sequence flanking the gRNA sequence (red) is shown in gray. POLL −/− clone # T2 was generated by CRISPR-Cas9 engineering and carries 7 bp deletion in both alleles. Sequence of the region harboring the 7 bp deletion is indicated in blue. c. Bar plot showing relative GFP following overexpression of indicated plasmids and co- transfection of left and right MMEJ reporter DNA constructs in HEK293T cells. GFP+ frequencies are normalized to transfection efficiency. Data represent mean. n = 1 experiment with triplicates for each condition, +/− s.e.m. Bottom panel: Immunoblot showing abundance of protein. d. gRNA sequence used to generate LIG4 −/− HEK293T cells (top) and XRCC4 −/− HEK293T cells (bottom) via CRISPR-Cas9 engineering. Schematic representation of human Lig4 (top) and Xrcc4 (bottom) with protein domains as well as location of gRNA sequence is indicated (red). e. Same as in Fig. 3f in XRCC4−/− HCT116 cells. Data represent mean. n = 1 experiment with triplicate for each condition, +/− s.e.m. Bottom panel: Immunoblot showing abundance of protein. f. Western blot of Polλ (top) and Gapdh (bottom) following transfection of either Polλ siRNA or siControl in DLD1 BRCA2+/+ (left) and DLD1 BRCA2 −/− cells (right).

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Quantitative RT-PCR, Expressing, Control, Sequencing, CRISPR, Generated, Over Expression, Cotransfection, Construct, Transfection, Western Blot

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a, A photocleavable nucleotide (pink) was converted into a nucleotide gap within the Polλ-DNA crystal via UV light, resulting in a MMEJ synapse bridged by a single G-C base pair. T, template strand; P, primer strand; D, downstream strand. b, Structure of Polλ:MMEJ synapse with incoming nucleotide. Polymerase subdomains, lyase (magenta), fingers (blue), palm (green) and thumb (purple), are shown as cartoon. DNA (purple, template strand; cyan, primer strand; magenta, downstream strand) and TTP are shown in stick representation. Atomic volume for the DNA is shown as a transparent white surface. c, Overall DSB substrate conformation. Microhomology is outlined with a black rectangle. TTP and DNA bases are shown in stick representation with the phosphate backbone as cartoon. Orange spheres, catalytic (Mec) and nucleotide (Nac) metals. Atomic volume is shown as white transparent surface. d, Downstream 5′-phosphate coordination of the polymerase-bound microhomology. Sidechains (yellow) and downstream DNA (magenta) are shown in stick representation. Lyase domain sidechain 5′ phosphate coordination is shown with black dashes; distances (Å) are indicated. Sidechains are shown in yellow stick representation. Blue sphere, water molecule. e, Template strand gap stabilization. Stabilizing sidechain interactions with the template marginal phosphates are shown with black dashes. A curved arrow indicates alternate conformations of the base of the primer strand nucleotide opposite the template strand gap. f, Key active site distances and metal coordination. Coordination (black) and key distances (red) are indicated with dashes. g, Active site conformation is consistent with catalysis. Simulated annealing (Fo−Fc) density is shown for TTP, primer terminal (Pn) nucleotide and active site metal atoms. Helix N that stacks with the incoming nucleotide is shown as a yellow cartoon.

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques:

Journal: Nature structural & molecular biology

Article Title: Polλ promotes microhomology-mediated end-joining

doi: 10.1038/s41594-022-00895-4

Figure Lengend Snippet: a. Structural comparison of Polλ bound to a microhomology-mediated DNA synapse and a single nucleotide gap (PDB id 2PFO). Differences (0−4.2 Å) in backbone Cα positioning are displayed as a heatmap colored from blue (0 Å) to white (0.5 Å) to red (1+Å) mapped onto the structure of the double strand break bound pol λ in cartoon representation. b. Overlay of the microhomology substrate containing gaps in template and primer strands with a single nucleotide gap substrate (PDB id 2PFO, transparent gray). Incoming TTP (green) or dUMPNPP (transparent gray) and downstream (magenta), primer (cyan), template (purple) strands are shown in stick representation. The orange spheres are active site metal ions and the purple sphere is a sodium atom. c. Template strand interactions in DSB bound Polλ. Template strand (magenta) and sidechains (yellow) are shown in stick representation. Key interactions are shown with black (side chains) or green (water) dashes. Waters are shown as blue spheres. Inset. Comparison of template strand positioning and gap marginal nucleotide interactions in structures of Polλ with a nick in the template strand (white transparent sticks, PDB id 7M0D31) and a gap in the same position (yellow sidechains, purple DNA). d. Differences in downstream 5′ phosphate coordination overlaid with the structure of a single nucleotide gap (PDB id 2PFO, transparent gray). e. Lyase domain and downstream primer shift compared to the structure with a single nucleotide gap (PDB id 2PFO). Protein backbone and DNA are shown in magenta cartoon and stick representation, respectively. f. Metal coordination in the active site. Shown is an overlay with a structure of Polλ bound to a single nucleotide gap and a non-hydrolyzable nucleotide (PDB id 2PFO, transparent gray).

Article Snippet: Polλ was detected using DNA Polλ rabbit polyclonal antibody (Novus Bio, catalog no. NB100-81665) diluted 1:1,000.

Techniques: Activity Assay, Comparison

Journal: Mutagenesis

Article Title: Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

doi: 10.1093/mutage/gey024

Figure Lengend Snippet: Fig. 2. Effects of the WRN and DNA pol λ double knockdown in U2OS cells on the mutant frequency induced by GO. Open columns, control plasmid containing G at position 122; closed columns, plasmid containing GO at position 122. Transfection experiments were performed six times. Data are expressed as the means + standard errors. *P < 0.05 vs. control RNA (Student’s t-test).

Article Snippet: To detect DNA pol λ, the membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and then incubated with a rabbit anti-DNA pol λ antibody (Bethyl Laboratories, Montgomery, TX, USA, catalogue no. A301-640A) in PBS-T containing 5% Blocking One overnight at 4°C.

Techniques: Knockdown, Mutagenesis, Control, Plasmid Preparation, Transfection

Journal: Mutagenesis

Article Title: Mutations induced by 8-oxo-7,8-dihydroguanine in WRN- and DNA polymerase λ-double knockdown cells.

doi: 10.1093/mutage/gey024

Figure Lengend Snippet: Fig. 1. Knockdowns of WRN and DNA pol λ by siRNAs. U2OS cells were treated with the siRNAs, and total protein was extracted at 24, 48 and 72 h after siRNA introduction. WRN and DNA pol λ expressions were analysed by western blotting.

Article Snippet: To detect DNA pol λ, the membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and then incubated with a rabbit anti-DNA pol λ antibody (Bethyl Laboratories, Montgomery, TX, USA, catalogue no. A301-640A) in PBS-T containing 5% Blocking One overnight at 4°C.

Techniques: Western Blot

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 3 Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG- Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Irradiation, Isolation, SDS Page, Immunoprecipitation, Incubation, Mutagenesis, Transfection, Control

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 4 Interaction of PAXX and XLF with Pol λ requires C-terminal Ku-binding regions. a EMSA showing that interaction of PAXX with Pol λ requires DNA- bound Ku. Reactions were performed with 10 or 20 nM IRDye® 700-labelled 5nt-gapped dsDNA (90-mer) and the following concentrations of FLAG- Ku70/80 (20 nM), FLAG-Pol λ (40 nM) or cleaved PAXX (100 nM). b Binding of PAXX to DNA-bound Ku requires C-terminal residues V199 and F201 of PAXX. Reactions were performed with 20 nM IRDye® 700-labelled 5nt-gapped dsDNA (90-mer) and the indicated concentrations of FLAG-PAXX-WT or a FLAG-PAXX-V199A/F201A mutant and FLAG-Ku70/80 (20 nM). c As described in Panel A, except that reactions contained FLAG-Ku (20 nM), FLAG-Pol λ (100 nM), FLAG-PAXX-WT (2.5 μM, left panel) or a V199A/F201A mutant (2.5 μM, right panel). d As described in Panel A, except that reactions contained FLAG-Ku (20 nM), FLAG-Pol λ (200 nM), FLAG-XLF-WT (2.5 μM, left panel) or a C-terminal FLAG-XLF (aa1-233) deletion mutant (2.5 μM, right panel)

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Binding Assay, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 5 Role of XRCC4 family members in the recruitment of Pol λ to laser microirradiation-induced DNA damage sites. a Upper, Schematic figure showing N-terminal EGFP- and mCherry-Pol λ fusion proteins; Lower, Representative immunofluorescence images showing that N-terminal EGFP- and mCherry-Pol λ fusion proteins are localised to nuclei in U2OS cells. b Recruitment of N-terminal EGFP-Pol λ to laser-induced DNA damage sites in U2OS cells. c Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS cells. Data shown are the mean and SEM from 16 individual cells. d Immunoblot analysis of N-EGFP-Pol λ expressing U2OS cells deficient in PAXX, XLF or XRCC4 generated by CRISPR-Cas9. WCL were resolved by SDS- PAGE and the indicated proteins detected by immunoblotting. e Localisation of N-mCherry-Pol λ in U2OS WT, PAXX-, XLF- and XRCC4-deficient cells. Representative immunofluorescence images showing that N-terminal mCherry-Pol λ fusion protein localises to nuclei in PAXX-, XLF- or XRCC4-deficient U2OS cells. Cells were co-stained with DAPI or dynamin-2, a perinuclear-enriched protein. f Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS-WT cells and cells deficient in PAXX, XLF or XRCC4. Data shown are the mean and SEM from WT, PAXX, XLF and XRCC4 knockout cells. Graphs shown are for the following cell numbers: WT: 8 cells; PAXX KO, 10 cells; XLF KO 17 cells; XRCC4 KO 18 cells. g Time course of N-EGFP-FLAG-Ku70 recruitment to laser-induced DNA damage sites in U2OS-WT cells and PAXX KO cells. Data shown are the mean and SEM from WT and PAXX knockout cells. Graphs shown are for the following cell numbers: WT: 17 cells; PAXX KO, 19 cells

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Western Blot, Expressing, Generated, CRISPR, SDS Page, Staining, Knock-Out

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 6 XRCC4 family proteins stimulate gap filling synthesis activity of Pol λ. a Gap filling activity of Pol λ-WT and a catalytically inactive Pol λ-D427A/ D429A/D490A mutant. b PAXX, XLF and XRCC4 stimulate gap-filling synthesis activity of Pol λ with an IRDye® 700-labelled 5nt-gapped dsDNA (33- mer) substrate. c As described in Panel B, except that some reactions also contained either FLAG-PAXX or –XLF alone d Gap filling synthesis assays were performed as described in Panel B with Pol λ immunoprecipitated from RPE-1 PAXX+/+ or PAXX KO cells incubated with or without XLF or XRCC4 siRNA

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Mutagenesis, Immunoprecipitation, Incubation

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 7 Head domains of XRCC4 Family Proteins interact with and stimulate Pol λ-dependent gap filling synthesis. a Schematic representation of XRCC4 family proteins. b PAXX head domain, but not the CC-CTR region, stimulates gap filling synthesis activity of Pol λ. c Silver stain and immunoblot analysis of purified XRCC4 family protein head domains. d Head domain of XRCC4 family proteins stimulate Pol λ-dependent gap filling synthesis activity. e Far- Western blot analysis showing that Pol λ-WT interacts with the head domain of XRCC4 family proteins. f Stimulation of Pol λ-dependent gap filling synthesis activity by PAXX-WT but not PAXX-VF, a non-Ku binding C-terminal mutant. g PAXX head domain stimulates comparable gap filling synthesis activity of Pol λ-WT and –R57A/L60A

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Silver Staining, Western Blot, Far Western Blot, Binding Assay, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 8 The Pol λ 8 kDa domain is required for stimulation of Pol λ-dependent gap filling activity via interaction with the head domain of XRCC4 family proteins. a Schematic representation of N-terminal Pol λ deletion mutants. b PAXX, XLF and XRCC4 stimulate gap filling synthesis activity of ΔBRCT- and ΔBRCT-Ser-Pro-Pol λ but not ΔBRCT-Ser-Pro-8kDa-Pol λ with an IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) substrate. c PAXX head domain promotes gap filling synthesis activity of ΔBRCT- and ΔBRCT-Ser-Pro-Pol λ but not ΔBRCT-Ser-Pro-8kDa-Pol λ. d Far-Western blot analysis showing that the head domain of XRCC4 family proteins interacts with Pol λ-WT and -ΔBRCT but not -ΔBRCT-Ser-Pro-8kDa

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Activity Assay, Far Western Blot

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 9 PAXX and XLF together with Pol λ to promote ligation of noncohesive DNA ends which requires gap filling activity of Pol λ. a–e Linear DNA substrates as shown were incubated with the indicated combinations of XRCC4/Lig IV, Ku70/80, PAXX, XLF and Pol λ and the joining efficiency quantified by qPCR with a TaqMan probe using a standard curve of log10 % joining efficiency versus Ct value generated using prejoined DNA fragments. DNA substrates were as follows: (a) EcoRV-PvuI blunt-2nt 3’ overhang; (b) EcoRV-KpnI blunt-4nt 3′ overhang; (c) EcoRV-EcoRV blunt-blunt ends; (d) EcoRI- KpnI 4nt 5’ overhang-4nt 3′ overhang, (e) EcoRV-BstEII blunt-5nt 5′ overhang; (f) As described in Panel (e), except that ligation assays contained either Pol λ-WT or a catalytically inactive Pol λ-3D mutant. Results shown are the mean ± SEM from 2–3 experiments performed in triplicate

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Ligation, Activity Assay, Incubation, Generated, Mutagenesis

Journal: Nature communications

Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair.

doi: 10.1038/s41467-018-06127-y

Figure Lengend Snippet: Fig. 10 Pol λ, PAXX and XLF function in common and parallel pathways. a Immunoblot analysis of control- or Pol λ siRNA-depleted U2OS PAXX KO, XLF KO and PAXX/XLF DKO cells. WCL were resolved by SDS-PAGE and indicated proteins detected by immunoblotting. b Clonogenic survival assays following IR (0-4 Gy) for U2OS WT, -PAXX KO, -XLF KO and -PAXX/XLF DKO cells with or without depletion of Pol λ Mean and SD from three independent experiments are shown. Statistical analysis was performed using a two-tailed paired t-test to compare cells incubated with Pol λ siRNA with control siRNA: 1 Gy - WT p = 0.91, PAXX KO p = 0.38, XLF KO p = 0.10, PAXX/XLF DKO p = 0.10; 2 Gy - WT p = 0.0003, PAXX KO p = 0.0007, XLF KO p = 0.36, PAXX/XLF DKO p = 0.82; 4 Gy - WT = 0.02, PAXX KO = 0.002, XLF KO p = not determined, PAXX/XLF DKO p = not determined. c Cartoon showing a model for regulation of Pol λ by XRCC4 family proteins. At DSBs that are positioned proximal to a Pol λ substrate gap XRCC4 family proteins strongly interact with Ku heterodimers via their C-terminal regions; their head domains promote gap filling synthesis activity via comparatively weakly binding to the 8 kDa domain of Pol λ, which interacts with the 5′ end of the gap. Pol λ strongly interacts with Ku heterodimers via its N-terminal BRCT domain

Article Snippet: Assays were also performed with endogenous Pol λ immunoprecipitated from RPE-1 cells using anti-rabbit Pol λ (Bethyl A301-640A).

Techniques: Western Blot, Control, SDS Page, Two Tailed Test, Incubation, Activity Assay, Binding Assay