Journal: Scientific Reports

Article Title: Bioorthogonal protein-DNA conjugation methods for force spectroscopy

doi: 10.1038/s41598-019-49843-1

Figure Lengend Snippet: Overview of the bio-orthogonal reactions, unnatural amino acids, and DNA modifications employed in this study. ( a ) Terminal alkynes ( 1 ) and azides ( 2 ) can form 1,4-disubstituted 1,2,3-triazoles in a 1,2-dipolar cycloaddition catalysed by copper. Azides can also react with strained alkynes such as DIBO/DBCO ( 3 ) in strain-promoted azide-alkyne cycloadditions that do not require a catalyst. Cyclopropenes ( 4 ) react with tetrazines ( 5 ) in inverse-electron-demand Diels-Alder reactions to diazanorcaradienes. ( b ) Lysine derivatives bearing alkyne, azide and cyclopropene moeities were introduced into PR65. ( c ) DNA oligonucleotides were modified with DBCO and tetrazine functional groups.

Article Snippet: Functionalised DNA handles of approximately 600 bp length were amplified from λ -DNA (e.g. Jena Bioscience) using the a triple biotinylated primer, a triple digoxigenin modified primer and a primer with a stable abasic site (Table , Metabion).

Techniques: Modification, Functional Assay

Journal: Scientific Reports

Article Title: Bioorthogonal protein-DNA conjugation methods for force spectroscopy

doi: 10.1038/s41598-019-49843-1

Figure Lengend Snippet: Attachment sites and SDS-PAGE of protein-oligo conjugations. ( a ) End-to-end attachment is achieved by substituting D5 and L588 with unnatural amino acids. When azides or cyclopropenes are incorporated and reacted with DBCO- or tetrazine-modified DNA oligos, reaction products can be visualised as larger molecular weight bands on an Coomassie stained SDS-PAGE gel. ( b ) Sfp-synthase mediated attachment and SPAAC are combined to exert forces on only the N-terminal half of the protein. Here, the first gel shows the reaction after 2 hours of incubation while the second shows the reaction containing both DNA oligos after overnight incubation. Un-cropped gels are available in the Supplementary Information (Figs and ).

Article Snippet: Functionalised DNA handles of approximately 600 bp length were amplified from λ -DNA (e.g. Jena Bioscience) using the a triple biotinylated primer, a triple digoxigenin modified primer and a primer with a stable abasic site (Table , Metabion).

Techniques: SDS Page, Modification, Molecular Weight, Staining, Incubation

Journal: Scientific Reports

Article Title: Bioorthogonal protein-DNA conjugation methods for force spectroscopy

doi: 10.1038/s41598-019-49843-1

Figure Lengend Snippet: Site-specific attachment of DNA handles to a protein is necessary for force spectroscopy using a double-dumbbell optical tweezers set up. A small DNA oligo is covalently linked to the protein and hybridized to single-stranded overhangs in the DNA handles. The trapped beads can then be moved apart to exert a force on the protein.

Article Snippet: Functionalised DNA handles of approximately 600 bp length were amplified from λ -DNA (e.g. Jena Bioscience) using the a triple biotinylated primer, a triple digoxigenin modified primer and a primer with a stable abasic site (Table , Metabion).

Techniques: Force Spectroscopy

Journal: Scientific Reports

Article Title: Bioorthogonal protein-DNA conjugation methods for force spectroscopy

doi: 10.1038/s41598-019-49843-1

Figure Lengend Snippet: DNA handle hybridization to SPAAC, IE-DA and Sfp-mediated protein-oligo conjugations purified using an S200 10/300 GL column and detected by agarose gel electrophoresis. A protein with two DNA oligos attached can hybridize with one or two double stranded DNA handles. The amount of protein added to achieve maximal hybridisation with two handles is optimized by titrating protein while the DNA amount is kept constant. Un-cropped gels are available in the Supplementary Information (Fig. ).

Article Snippet: Functionalised DNA handles of approximately 600 bp length were amplified from λ -DNA (e.g. Jena Bioscience) using the a triple biotinylated primer, a triple digoxigenin modified primer and a primer with a stable abasic site (Table , Metabion).

Techniques: Hybridization, Purification, Agarose Gel Electrophoresis

Journal: Scientific Reports

Article Title: Bioorthogonal protein-DNA conjugation methods for force spectroscopy

doi: 10.1038/s41598-019-49843-1

Figure Lengend Snippet: Representative full-length force-extension traces of all attachments. As a force is applied to the DNA-protein construct, the more pliable DNA stretches first before the protein experiences any force and unfolds in a series of force peaks combined with transitions that cannot be described with current polymer models. Unfolding and refolding traces are coloured in darker and lighter shades, respectively. All traces were obtained at pulling speeds of 10 nm s −1 . See Figs and for multiple pulling traces of the same molecule.

Article Snippet: Functionalised DNA handles of approximately 600 bp length were amplified from λ -DNA (e.g. Jena Bioscience) using the a triple biotinylated primer, a triple digoxigenin modified primer and a primer with a stable abasic site (Table , Metabion).

Techniques: Construct, Polymer

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: For overexpression of Polλ WT, 1 × 10 4 HEK293T cells were plated and, after 24 h, 200 ng either Polλ WT-Myc-DDK-tagged (Origene catalog no. RC230157) or Empty vector control plasmid was transfected using lipofectamine 2000.

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: For overexpression of Polλ WT, 1 × 10 4 HEK293T cells were plated and, after 24 h, 200 ng either Polλ WT-Myc-DDK-tagged (Origene catalog no. RC230157) or Empty vector control plasmid was transfected using lipofectamine 2000.

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

Journal: Journal of Cell Science

Article Title: γ-tubulin mediates DNA double-strand break repair

doi: 10.1242/jcs.262255

Figure Lengend Snippet: γ-tubulin associates with DSB-mimicking DNA in vitro , and is recruited to DNA damage sites in cells. (A) Pulldown with biotin-labeled dsDNA (500 bp with free ends, as described in ) mimicking double-strand breaks (DSBs) was performed using HeLa cell lysates. A control (ctr) pulldown was performed using blank beads. The input, control pulldown and dsDNA pulldown samples were analyzed by immunoblotting (IB). n =3 independent experiments. (B) SCC38 cells were laser-microirradiated and analyzed by immunofluorescence for γ-tubulin and phospho-ATM S1981, as described in the Materials and Methods. The images were taken 3 min post laser treatment. The region of laser microirradiation is denoted by the white arrow. n =3 independent experiments. (C,D) HeLa cells with transient expression of mCherry–γ-tubulin and GFP–polymerase λ were laser microirradiated after pre-sensitization and imaged, as described in the Materials and Methods. The images were taken 5 min post laser treatment. Colocalization of mCherry–γ-tubulin and GFP–polymerase λ after laser microirradiation was quantified in panel C and shown in panel D. Arrows in the inset images in C indicate the points chosen for colocalization analysis. The regions of laser microirradiation are denoted by white arrows in D. n =3 independent experiments. (E) HeLa cells were treated with etoposide (1 µM, 10 min) and analyzed by immunofluorescence for γ-tubulin and 53BP1. The colocalization is shown. n =3 independent experiments. (F) Whole-cell extracts (WCE) and nuclear fractions were isolated from HeLa cells with or without etoposide treatment (1 µM, 3 h), as described in the Materials and Methods, and analyzed by IB for γ-tubulin and histone H3. (G) The IB results in panel F were quantified. Band intensities of γ-tubulin were normalized to those of histone H3. n =3 independent experiments. Bars show mean±s.d. Two-tailed unpaired Student's t -test was performed; ns, not significant; * P <0.05.

Article Snippet: SDS-PAGE and immunoblotting were carried out using the following antibodies: anti-artemis (D7O8V, #13381, 1:500), anti-Ku80 (C48E7, #2180, 1:1000), anti-RAD51 (D4B10, #8875, 1:800), anti-DNA-PKcs (E6U3A, #38168, 1:1000), anti-Ku70 (D10A7, #4588, 1:1000), anti-RPA70 (#2267, 1:1000), anti-DNA ligase IV (D5N5N, #14649, 1:1000), anti pATM/ATR substrate (#6966, 1:1000) and anti-XLF (#2854, 1:1000) from Cell Signaling Technology (Beverly, MA, USA); anti-α-tubulin (ab7291, 1:1000) and anti-γ-tubulin (ab11317, 1:1000) from Abcam (Cambridge, MA, USA); anti-γ-H2AX (Ser 139) (sc-517348, 1:1000), anti-total H2AX (sc-517336, 1:1000), anti-PARP1 (B-10, sc-74470, 1:1000), anti-histone H3 (1G1, sc-517576, 1:1000), anti-DNA polymerase λ (E-11, sc-373844, 1:1000), anti-RPA32 (9H8, sc-56770, 1:1000) and anti-RPA70 (H-7, sc-48425, 1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: In Vitro, Labeling, Control, Western Blot, Immunofluorescence, Expressing, Isolation, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Decellularized sciatic nerve matrix as a biodegradable conduit for peripheral nerve regeneration

doi: 10.4103/1673-5374.237126

Figure Lengend Snippet: DNA assay and hematoxylin and eosin staining of decellularized sciatic nerve. DNA assays were performed to confirm decellularization (A). * P = 0.05. In addition, cells of nerve tissue (arrows) were examined by hematoxylin and eosin staining. Nuclei of cells (arrows) were present in the tissues before decellularization (B, C), but no cells were observed in the tissues after decellularization (D, E). Scale bars: 500 µm in B and D, 100 µm in C and E.

Article Snippet: Hemotoxylin and eosin (H&E) tissue staining (Cat No. 3801698, Leica, Richmend, IL, USA) and DNA quantification (Cat No.1660001EDU, Bio-RAD, Hercules, California, USA) were performed to analyze the decellularization efficiency of porcine nerve tissue.

Techniques: Staining