1. Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining 
Pablo De Ioannes, Shruti Malu, Patricia Cortes, Aneel K. Aggarwal 
Cell Reports 
Volume 2, Issue 6, Pages (December 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1505-1512DOI: (10.1016/j.celrep.2012.11.004)
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3. Figure 1 Crystal Structure of LigIV-DBD apo Form
(A) Ribbon diagram of LigIV-DBD. Secondary structure elements and important amino acids side chains are labeled.
(B) Structural alignment of human Lig-DBDs. LigIV-DBD superimposed on LigI-DBD (PDB ID code 1X9N, residues 284 to 531) and LigIII-DBD (PDB ID code 3L2P, residues 168 to 375).
(C) Electrostatic surface representation of LigIV-DBD colored, with saturating blue and red at +7 kT/e and −7 kT/e, respectively. A relatively uncharged and exposed surface area on LigIV-DBD is highlighted by a dashed oval. Important amino acids are labeled.
See also Figure S1.
Cell Reports 2012 2, DOI: ( /j.celrep )
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4. Figure 2 Crystal Structure of LigIV-DBD/cArt-Pep Complex
(A) A schematic of LigIV and Artemis domain organization and of regions that interact.
(B) Overall structure of the LigIV-DBD/cArt-Pep complex. LigIV-DBD (green) is represented in ribbon and cArt-Pep (pink) is represented in stick. Simulated annealing 2Fo-Fc omit electron density is also shown for cArt-Pep, contoured at σ = 1.0. The axis of helix α2 is marked by two straight lines to indicate the bending.
(C) LigIV-DBD and cArt-Pep displayed separately to highlight the important amino acids.
(D) Electrostatic potential surface of LigIV-DBD and cArt-Pep when displayed separately.
(E) Superposition of apo (red) and peptide bound (green) forms of LigIV-DBD to show the movements in helix α2 (∼9 Å) and residue F49. The peptide was omitted for clarity.
(F) Detailed view of LigIV-DBD/cArt-Pep interaction surface. cArt-Pep and LigIV-DBD are shown in pink and green, respectively. The molecular surface of LigIV-DBD is presented in gray. Hydrogen bonds are depicted in dashed yellow lines.
See also Figure S2.
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5. Figure 3 LigIV-Artemis Interaction
(A–F) ITC titration profile of cArt-Pep with LigIV-DBD WT (A), LigIV-DBD F42A (B), LigIV-DBD F49A (C), LigIV-DBD F42AF49A (D), LigIV-DBD D18H (E), and LigIV-DBD V14A (F).
(G) ITC profile for cArt-Pep dilution.
(H) Coimmunoprecipitation experiments with full-length and mutant LigIV. Flag IP was performed on lysates of 293T cells transfected with FNT (Flag-NLS-Thioredoxin), Flag Ligase IV (FLLIV), or its point mutants FL42A and FL49A with untagged Artemis. Western blot analysis for Flag, Artemis, and endogenous XRCC4 was performed. Levels of Artemis and XRCC4 in total cell extract (TCE) are shown as controls.
See also Figure S3.
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6. Figure 4 Comparison of LigIV-DBD to LigI and LigIII
(A) Fluorescent anisotropy isothermal binding curves for cArt-Pep against LigIV-DBD, LigI-DBD, and LigIII-DBD. cArt-Pep binds specifically to LigIV-DBD. Because of low affinity, LigI-DBD and LigIII-DBD could not be fully titrated against cArt-Pep and the Kds were not determined (ND).
(B) Close up views of LigIV-DBD/cArt-Pep binding (left panel) and models of cArt-Pep docked against LigI-DBD (center panel) and LigIII-DBD (right panel). Note the steric overlap between cArt-Pep and the N-terminal region of LigI-DBD.
(C) A model of LigIV catalytic core bound to cArt-Pep (pink) and DNA substrate (orange). LigIV-DBD (green) and catalytic core (NTase and OBD) (light purple) are represented as surfaces. The peptide binding site (yellow) and the DNA binding site are on opposite surfaces. A schematic of LigIV domain organization is shown at the bottom.
See also Figure S4.
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7. Figure S1
Apo LigIV-DBD Fitted to the Initial Experimental Electron Density Map and Topology Diagram, Related to Figure 1
(A) Anomalous difference Fo-Fc map (selenium sites, purple) and the initial modified 2Fo-Fc experimental electron density for apo LigIV-DBD (gray) contoured at σ = 7 and σ = 1, respectively. Selenomethionine side chains are depicted as sticks.
(B) Topology diagram of apo LigIV-DBD. Helices are labeled according to Figure 1A. Compositions of helical subdomains are indicated under brackets.
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8. Figure S2 Structural Comparison of Lig-DBDs, Related to Figure 2
Structural and electrostatic comparisons between mammalian Lig-DBDs: LigI-DBD (A, D, G), LigIII-DBD (B, E, H), and LigIV-DBD (C, F, I). The superimposed DNA substrates are depicted in stick. Electrostatic surfaces are represented with saturating blue and red at +7 kT/e and −7 kT/e, respectively.
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9. Figure S3
Secondary Structure Alignment of Lig-DBDs, Related to Figure 3
Structural based multiple sequence alignment of Lig-DBDs. The structures are as follow: hLigIV (human Ligase IV, this report), hLigI (human ligase I, PDB id 1X9N), hLigIII (human Ligase III, PDB id 3L2P), AfulLig (A. fulgidu DNA ligase, PDB id 3GDE), pfurlLig (P. furiosus DNA ligase, PDB id 2CFM), ssolLig (S. Solfataricus DNA ligase PDB id 2HIV). The degree of sequence identity is represented in colors from light to dark blue. The secondary structure of LigIV is shown on top of the alignment. The LigIV residues important for cArt-Pep binding are highlighted in green.
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10. Figure S4 A Model of LigIV/Artemis/XRCC4 Complex, Related to Figure 4
The model is based on the LigIV/cArt-Pep structure (green and wheat), a homology model of the LigIV catalytic domain (light blue) based on the LigI crystal structure (PDB id 1X9N) (Pascal et al., 2004), the XRCC4/BRCT structure (yellow and red; PDB id 3II6) (Wu et al., 2009), and a homology model of the Artemis catalytic core (β-Lact/β-CASP) (orange) based on the CPSF-73 crystal structure (PDB id 2I7T) (Mandel et al., 2006). The substructures match the arrangement in an EM model of LigIV/XRCC4 complex (Recuero-Checa et al., 2009). The linkers connecting the substructures are depicted by dashed lines. The bottom panel shows the domain organization of Artemis, LigIV, and XRCC4.
Cell Reports 2012 2, DOI: ( /j.celrep )
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