pgbkt7 brct  (TaKaRa)

Journal: NPJ Genomic Medicine

Article Title: Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

doi: 10.1038/npjgenmed.2016.1

Figure Lengend Snippet: BRCA1 variant mapping. ( a ) Overview of BRCA1 variants from amino acid residues 1396–1863. ( b ) Depiction of BRCA1 protein domains and motifs in the aa 1,315–1,863 region and the variants that have been tested by transcriptional assays in previous (blue) or the current (red) studies ( c ) Depiction of BRCA1 constructs used in this study and the locations of the variants tested. ( d ) VarCall analysis of missense variants in the carboxy-terminal region (aa 1,315–1,863 of the BRCA1 protein). Transcriptional assays were performed for 98 missense variants in the aa 1,395–1,864 context (9 of these represent retests), 10 missense variants were tested by transcriptional assays in the aa 1,315–1,863 context (3 variants found in the population, 7 variants not currently known to occur in the population). Transcriptional assays were performed using a luciferase reporter system where 293T cells were co-transfected with a reporter plasmid, pG5Luc, which contains a firefly luciferase gene under the control of five GAL4 binding sites, a pCDNA3 plasmid coding for either wild-type or variant BRCA1 constructs fused to the GAL4 DNA-binding domain, and an internal control containing a Renilla luciferase gene constitutively expressed (described in Carvalho et al. (2009)) . Results of these transcriptional assays were analysed using VarCall to predict the likelihood of pathogenicity. In addition, the coiled-coil domain and secondary structures of the BRCT domain were overlaid on the results. See for additional details.

Article Snippet: Fragments coding for the tandem BRCT domains of MCPH1 (aa 649–832) and MDC1 (aa 1,894–2,079) were obtained by PCR amplification ( ) and cloned into the pGBKT7 or pGADT7 vectors (Clontech) as fusions to the GAL4 DBD or AD, respectively. pGBKT7 BRCT, pGADT7 BRCT or empty pGBKT7 were transformed in the Y2HGold Saccharomyces cerevisiae strain and plated on dropout medium lacking Tryptophan (SD-Trp) or Leucine (-Leu) and number of colonies was scored.

Techniques: Variant Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

doi: 10.1038/npjgenmed.2016.1

Figure Lengend Snippet: Mutagenesis screen to identify loss of function missense variants in MCPH1 and MDC1 tandem BRCT domains. ( a ) Expression in yeast of proteins containing the tandem BRCT of MCPH1 or MDC1 fused to the GAL4 DNA-binding domain (DBD), but not to the activation domain (AD) of GAL4, lead to growth inhibition and a small colony phenotype. Libraries of mutagenised constructs coding for the tandem BRCTs of MCPH1 or MDC1 were transformed into yeast and transformants with regular size colonies (carrying loss of function mutants) were isolated and sequenced to identify residues that disrupt the BRCT function. ( b ) Loss of function variants (red) cluster around the BRCT phosphopeptide-binding pocket in MCPH1 (PDB ID: 3U3Z) and MDC1 (PDB ID: 2AZM). ( c ) MCPH1 BRCT variants found in cancer samples and predicted to impair function, as inferred by BRCA1 data sets, (R693H and W815R) abrogate the small-colony phenotype, whereas a predicted neutral mutation (N661S) does not.

Article Snippet: Fragments coding for the tandem BRCT domains of MCPH1 (aa 649–832) and MDC1 (aa 1,894–2,079) were obtained by PCR amplification ( ) and cloned into the pGBKT7 or pGADT7 vectors (Clontech) as fusions to the GAL4 DBD or AD, respectively. pGBKT7 BRCT, pGADT7 BRCT or empty pGBKT7 were transformed in the Y2HGold Saccharomyces cerevisiae strain and plated on dropout medium lacking Tryptophan (SD-Trp) or Leucine (-Leu) and number of colonies was scored.

Techniques: Mutagenesis, Expressing, Binding Assay, Activation Assay, Inhibition, Construct, Transformation Assay, Isolation

Journal: Nucleic Acids Research

Article Title: Yeast two-hybrid junk sequences contain selected linear motifs

doi: 10.1093/nar/gkr600

Figure Lengend Snippet: Fitted MLE parameters demonstrate predicted properties of motifs under selection in seven Y2H screens

Article Snippet: pGBKT7-LIG4-tBRCT (tandem BRCT domains) was co-transformed into yeast strain MaV203 (Invitrogen) along with pACT2-Empty, pACT2-PA2G4 wild-type (WT), or pACT2-PA2G4 6KA mutant (in which all six lysines were mutated to alanines).

Techniques: Selection

Journal: Nucleic Acids Research

Article Title: Yeast two-hybrid junk sequences contain selected linear motifs

doi: 10.1093/nar/gkr600

Figure Lengend Snippet: Proposed binding mechanism for the LIG4 BRCT tandem domains and PA2G4 as mediated by the SLiM KKKKKK . ( A ) LIG4 BRCT tandem domains (PDB 3ii6) with the linker region in magenta. ( B ) LIG4 BRCT tandem domains (PDB 3ii6) bound to coiled coil of XRCC4 (in cyan). Lysine residues are shown as orange sticks. Two lysines in XRCC4 (K187, K188) are critical to the binding interaction. If a similar mechanism is involved in the interaction of LIG4 and PA2G4, it would likely require a PA2G4 dimer, since both helices in the XRCC4 coiled-coil contain lysine rich regions. Figures created with PyMOL.

Article Snippet: pGBKT7-LIG4-tBRCT (tandem BRCT domains) was co-transformed into yeast strain MaV203 (Invitrogen) along with pACT2-Empty, pACT2-PA2G4 wild-type (WT), or pACT2-PA2G4 6KA mutant (in which all six lysines were mutated to alanines).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Yeast two-hybrid junk sequences contain selected linear motifs

doi: 10.1093/nar/gkr600

Figure Lengend Snippet: Y2H direct binding assay. Mutation of the KKKKKK SLiM abrogates binding to LIG4 tandem BRCT domains. Only cells lacking protein-protein interactions between bait and prey grow on this selection (-Leu, -Trp containing 0.2% 5FOA plates) because 5FOA is converted to the genotoxic 5FU when Ura is activated in cells containing the interaction. Empty vector and wild-type PA2G4 vector shown as controls. Cells were plated in serial dilutions of a saturated liquid culture.

Article Snippet: pGBKT7-LIG4-tBRCT (tandem BRCT domains) was co-transformed into yeast strain MaV203 (Invitrogen) along with pACT2-Empty, pACT2-PA2G4 wild-type (WT), or pACT2-PA2G4 6KA mutant (in which all six lysines were mutated to alanines).

Techniques: Binding Assay, Mutagenesis, Selection, Plasmid Preparation