Journal: DNA repair

Article Title: Ligand binding characteristics of the Ku80 von Willebrand domain

doi: 10.1016/j.dnarep.2019.102739

Figure Lengend Snippet: Selected crystallographic data.

Article Snippet: Crystallization, data collection and structure determination The human Ku-binding motif (KBM) peptides from APLF, MRI, and WRN (KBM APLF , KBM MRI , and KBM WRN ) and the KBMX peptide from XLF (KBMX XLF ) were obtained from GenScript.

Techniques:

Journal: DNA repair

Article Title: Ligand binding characteristics of the Ku80 von Willebrand domain

doi: 10.1016/j.dnarep.2019.102739

Figure Lengend Snippet: Human and Xenopus KBM sequences

Article Snippet: Crystallization, data collection and structure determination The human Ku-binding motif (KBM) peptides from APLF, MRI, and WRN (KBM APLF , KBM MRI , and KBM WRN ) and the KBMX peptide from XLF (KBMX XLF ) were obtained from GenScript.

Techniques: Sequencing

Journal: DNA repair

Article Title: Ligand binding characteristics of the Ku80 von Willebrand domain

doi: 10.1016/j.dnarep.2019.102739

Figure Lengend Snippet: Hydrogen bonding between xlKu80 vWA (gray) and (A) APLF (cyan), (B) WRN (green), and (C) MRI KBM peptide (magenta) is indicated by black dotted lines. Interacting vWA residues are labeled, and each peptide residue type is indicated in red. In some cases, disordered sidechains have been truncated. Interpeptide H-bonds are shown as red dotted lines, and H-bonds mediated by water molecules are in blue.

Article Snippet: Crystallization, data collection and structure determination The human Ku-binding motif (KBM) peptides from APLF, MRI, and WRN (KBM APLF , KBM MRI , and KBM WRN ) and the KBMX peptide from XLF (KBMX XLF ) were obtained from GenScript.

Techniques: Labeling, Residue

Journal: Nucleic Acids Research

Article Title: Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

doi: 10.1093/nar/gkx941

Figure Lengend Snippet: Crystal structures of the APLF FHA domain unliganded and bound to a phosphorylated XRCC1 peptide. ( A ) Cartoon representation of apo-APLF FHA domain ( green ) with the 10 β-strands numbered. ( B ) Stereo view showing an overlay of α-carbon traces of APLF FHA ( green ) with the FHA domains of PNKP ( blue ) and APTX ( magenta ). The APLF FHA structures in complex with ( C ) XRCC1 pSpT-9 diphosphopeptide (protein, light gray ; peptide, blue ) and ( D ) XRCC1 EpT-9 monophosphopeptide ( tan , protein; pink , peptide) are represented. Simulated annealing Fo-Fc omit maps of each phosphopeptide ( green mesh ) contoured at 3.0 σ for the diphosphopeptide and 2.5 σ for the monophosphopeptide are displayed. The peptide residues are annotated in blue with underlined, italic residue names, and the APLF FHA residues important for binding are annotated in black . Hydrogen-bond interactions are also depicted ( red dotted line ). ( E ) The topologies of the β3-β4 and β5-β6 loops in the APLF FHA domain for the apo ( green ), the XRCC1 pSpT-9 diphosphopeptide-complexed ( light gray ), or the XRCC1 EpT-9 -monophosphopeptide-complexed ( tan ) structures are represented. Hydrogen-bonds that sustain a binding-ready conformation are indicated by black, red or cyan dashed lines, respectively.

Article Snippet: The FHA domain coding sequence (174–488 bp) of human APLF (APLF FHA), (NCBI reference sequence: NM_173545), with additional sequences at both ends for Gateway LR reaction, was synthesized and cloned into the pUC57 vector by GenScript.

Techniques: Phospho-proteomics, Residue, Binding Assay

Journal: Nucleic Acids Research

Article Title: Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

doi: 10.1093/nar/gkx941

Figure Lengend Snippet: 31 P NMR titration data for diphosphorylated XRCC1 and XRCC4 peptides as a function of the APLF FHA domain concentration. 31 P NMR spectra of samples containing ( A ) 0.5 mM of an 18-residue XRCC1 peptide phosphorylated on Ser518/Thr519 (XRCC1 pSpT-18 ), and ( B ) 0.5 mM of an 12-residue XRCC4 peptide phosphorylated on Ser232/Thr233 (XRCC4 pSpT-12 ) were titrated with the APLF FHA domain at the ratios indicated. The pSer resonance is indicated by a blue arrow, and the pThr resonance by a red dotted arrow. Titration studies were performed in 25 mM HEPES, 25 mM MES, 150 mM NaCl, 1 mM EDTA, pH 7.4 in the presence of a 0.25 mM TMP chemical shift reference. Resonances arising from impurities in the XRCC1 pSpT-18 sample are indicated with an X.

Article Snippet: The FHA domain coding sequence (174–488 bp) of human APLF (APLF FHA), (NCBI reference sequence: NM_173545), with additional sequences at both ends for Gateway LR reaction, was synthesized and cloned into the pUC57 vector by GenScript.

Techniques: Titration, Concentration Assay, Residue

Journal: Nucleic Acids Research

Article Title: Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

doi: 10.1093/nar/gkx941

Figure Lengend Snippet: Chemical shift perturbations vs. peptide length. ( A ) Amide chemical shift changes for 0.1 mM U-[ 15 N]APLF FHA domain in the presence of 0.8 mM XRCC1 pSpT-9 ( blue bars ) or XRCC1 pSpT-18 ( orange bars ). ( B ) 1 H, 15 N-HSQC spectra of U-[ 15 N]APLF FHA domain as a function of XRCC1 pSpT-9 ( left panel ) or XRCC1 pSpT-18 ( right panel ). Peptide concentrations (in μM): 0 ( red ), 10 ( orange ), 20 ( yellow ), 50 ( green ), 100 ( blue ), 200 ( purple ), 400 ( cyan ) and 800 ( pink ). Titration studies were performed in a NMR buffer containing 25 mM HEPES, 25 mM MES, 150 mM NaCl, 1 mM EDTA, pH 7.4.

Article Snippet: The FHA domain coding sequence (174–488 bp) of human APLF (APLF FHA), (NCBI reference sequence: NM_173545), with additional sequences at both ends for Gateway LR reaction, was synthesized and cloned into the pUC57 vector by GenScript.

Techniques: Titration

Journal: Nucleic Acids Research

Article Title: Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

doi: 10.1093/nar/gkx941

Figure Lengend Snippet: XRCC1 at the nexus of three different repair pathways. Schematic illustrating NHEJ backup recruitment by APLF. In addition to the standard abbreviations, KuBM—Ku binding motif; FBM—FHA domain binding motif. NHEJ proteins ( blue ), alt-NHEJ proteins ( orange ), proteins involved in multiple pathways ( gray ).

Article Snippet: The FHA domain coding sequence (174–488 bp) of human APLF (APLF FHA), (NCBI reference sequence: NM_173545), with additional sequences at both ends for Gateway LR reaction, was synthesized and cloned into the pUC57 vector by GenScript.

Techniques: Binding Assay