Journal: eLife

Article Title: Human RPA activates BLM’s bidirectional DNA unwinding from a nick

doi: 10.7554/eLife.54098

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pTWIN1 (plasmid) , New England BioLabs , N6951S , Expression of core BLM in E. coli.

Techniques: Recombinant, Lambda DNA Preparation, Plasmid Preparation, Expressing, DNA Purification, Staining, Software

Journal: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a An optical tweezers setup to study mitochondrial translation initiation. RNA construct containing the first 51 nucleotides of MT-CO2 mRNA was ligated to biotinylated lambda DNA, allowing the 5′-end accessible for mitoribosome binding. b In order to monitor both mitoribosomal subunits, mtLSU and mtSSU were labeled with Cy5 and Atto488, respectively, and initiation reactions were made by incubating either double-labeled monosome (b, 1.) or Cy5-labeled mtSSU (b, 3.) with the DNA/RNA hybrid, mtIF3, mtIF2 (GTP), and fMet-tRNA Met i . An RNA construct without start codon was used as a negative control (b, 2.). c Representative images showing binding of the mitoribosomal subunits tested by simultaneous dual-color confocal imaging. d Quantification of the binding events. The number of detected fluorescently labeled species were normalized to the number of trapped imaged DNA. Bars represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Construct, Lambda DNA Preparation, Binding Assay, Labeling, Negative Control, Imaging

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: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a Surface representation of the mtSSU with mtIF3 (orange) and mtIF2 (blue). Empty tRNA-binding sites and mRNA channel are indicated. The binding of mtIF2 is accomplished due to mtIF3 NTD restricting the mtSSU head movement. b Relative positions of mtIF3 colored by domains (NTD purple, linker dark gray, CTD orange, CTE red) and mtIF2.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a Two-color fluorescence cross-correlation spectroscopy analysis of the initiation complex components. The mtSSU was incubated with an excess of Cy3-fMet-tRNA fMet i , Cy5-mtIF3, mtIF2(GTP), and Atto390-mRNA, then purified by a sucrose gradient. Products may contain unlabeled mtIF2. b The functionality of mtPIC was validated by the ability to form a complete initiation complex when incubated with an excess of mtLSU, Cy3-fMet-tRNA fMet i , Atto390-mRNA, and unlabeled mtIF2 (GTP). After purification of the monosome, detection of both Cy3 and Atto390 fluorophores confirmed binding of mRNA and tRNA. Source data are provided as a Source Data file.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Fluorescence, Spectroscopy, Incubation, Purification, Binding Assay

Journal: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a Comparison of mtIF2 binding in mtPIC-2 with the complete initiation complex (PDB ID: 6GAW). Upon association of the mtLSU (blue), required for complete initiation complex, the mtIF2-domain IV moves towards the mtSSU. The conformational change is represented by the difference vectors for each C α atom of domain IV. For the other domains, the flexibility is represented through ribbon thickness according to B-factor. b Zoom-in panels showing contacts of the mtIF2-specific insert and linker domain with the mtSSU. c Schematic representation of the mtIF2 with the corresponding color-code to the conformational changes.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a An optical tweezers setup to study mitochondrial translation initiation. RNA construct containing the first 51 nucleotides of MT-CO2 mRNA was ligated to biotinylated lambda DNA, allowing the 5′-end accessible for mitoribosome binding. b In order to monitor both mitoribosomal subunits, mtLSU and mtSSU were labeled with Cy5 and Atto488, respectively, and initiation reactions were made by incubating either double-labeled monosome (b, 1.) or Cy5-labeled mtSSU (b, 3.) with the DNA/RNA hybrid, mtIF3, mtIF2 (GTP), and fMet-tRNA Met i . An RNA construct without start codon was used as a negative control (b, 2.). c Representative images showing binding of the mitoribosomal subunits tested by simultaneous dual-color confocal imaging. d Quantification of the binding events. The number of detected fluorescently labeled species were normalized to the number of trapped imaged DNA. Bars represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Construct, Lambda DNA Preparation, Binding Assay, Labeling, Negative Control, Imaging

Journal: Nature Communications

Article Title: Distinct pre-initiation steps in human mitochondrial translation

doi: 10.1038/s41467-020-16503-2

Figure Lengend Snippet: a Translation initiation in mammalian mitochondria. The various structures are summarized in the order that represents a possible initiation pathway in which mtIF2 binding precedes mtLSU and tRNA binding. In this pathway, mtIF3 and mS37 stabilize the mtSSU head for the accommodation of mtIF2. Joining of the mtLSU may result in the conformational change of mtIF2 and GTPase activation that leads to tRNA and mRNA accommodation. Alternatively, transient binding of both mRNA and tRNA (not detected with our techniques) to the mtSSU after mtIF3 departure precede recruitment of the mtLSU. b Translation initiation in bacteria. Binding of canonical mRNA, fMet-tRNA Met i and initiator factors precede subunit joining and formation of the complete initiation complex.

Article Snippet: A codon-optimized (Genscript) DNA construct corresponding to the mature form of human mtIF2 (amino acids 38–727) or human mtIF3 (amino acids 32–278) was cloned into a pET-24b vector (Novagen).

Techniques: Binding Assay, Activation Assay