1. Crystal Structure of Eukaryotic DNA Ligase–Adenylate Illuminates the Mechanism of Nick Sensing and Strand Joining 
Mark Odell, Verl Sriskanda, Stewart Shuman, Dimitar B. Nikolov 
Molecular Cell 
Volume 6, Issue 5, Pages (November 2000)
DOI: /S (00)

2. Figure 1
Structure-Based Alignment of Chlorella Virus and T7 DNA Ligases
The secondary structures of Chlorella virus ligase (ChV) and T7 ligase (T7) are shown above and below their respective amino acid sequences. The crystal structures were aligned using the program O. Gaps in the alignment are indicated by dashes. Peptide segments not visualized in the crystal structures are printed in red. Protease-sensitive sites within ChV ligase are denoted by green arrowheads. The conserved nucleotidyl transferase motifs are denoted above the ChV sequence; motifs I, III, IIIa, IV, and V are highlighted in yellow boxes.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2 Overall Fold of the Chlorella Virus DNA Ligase–Adenylate
The figure was prepared with the program SETOR. The N-terminal domain 1 is colored in purple; the C-terminal domain 2 (an OB fold) is colored in cyan.
(A) The lysyl–AMP adduct at the active site is shown.
(B) The ligase molecule is oriented to highlight a sulfate bound on the surface of domain 1.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 Electron Density at the Adenylate Binding Site
The figure shows the electron density surrounding the covalent lysyl–AMP adduct contoured at 1.2 σ.
(A) The MIR map.
(B) 2|Fo − Fc| omit map of the refined structure.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Remodeling of the Adenosine Binding Pocket after Ligase–Adenylate Formation and Structural Insights into Nick Recognition and Metal Binding
(A) Stereo view of the nucleoside binding pocket of ChV ligase highlighting interactions of motif I and III side chains with the ribose sugar of the lysyl–adenylate adduct. The ChV polypeptide backbone is colored purple, and the side chains are in CPK. The equivalent structural elements of the T7 ligase–ATP complex (colored all in green) are aligned to the ChV structure based on superimposition of the adenine base. Side chain–side chain and side chain–sugar contacts are denoted by dashed lines: blue for ChV ligase and yellow for T7 ligase.
(B) Stereo view highlighting the interactions of the AMP phosphate and the nearby sulfate on the surface of domain 1 (presumed to represent the 5′ PO4 at the nick). The position of the metal in the active site in the lutetium-soaked crystal is denoted by the cyan sphere.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
Comparison of the Chlorella Virus Ligase and Capping Enzyme Structures
Chlorella virus ligase–AMP (purple) versus capping enzyme–GMP (green). The structures were superimposed with reference to the nucleotide binding pocket of domain 1. The lysyl–AMP adduct and the sulfate in the ligase are shown. The guanylate and a sulfate are similarly positioned in the capping enzyme structure. The figure highlights the large movement of domain 2 from the closed state (capping enzyme) to a wide-open conformation (ligase) that exposes a DNA binding surface. The principal flexion points within the interdomain linkers (in motif V) are indicated by the short arrows.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6 Surface Topology and Electrostatics
The space-filling surface images of the Chlorella virus ligase–adenylate were prepared with the program GRASP. Positive surface charge potential is shown in blue, and negative potential in red. The sulfate is colored green and the AMP is yellow.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
Nick Joining and Nick Sensing by Mutated Versions of Chlorella Virus DNA Ligase
(A) Protein purification. Aliquots (2 μg) of the phosphocellulose fractions of wild-type ligase and the indicated mutant proteins were analyzed by SDS–PAGE. Polypeptides were visualized by staining the gel with Coomassie brilliant blue dye. The positions and sizes (in kDa) of marker proteins are indicated on the left.
(B) Mutational effects on nick joining. Ligation reaction mixtures contained 1 pmol of nicked duplex DNA substrate and 3.4 ng of the indicated phosphocellulose preparation of His-tagged ligase. The products were resolved by denaturing polyacrylamide gel electrophoresis. An autoradiogram of the gel is shown. The structure of the nicked DNA substrate is shown with the 5′ 32P label at the nick indicated by the dot.
(C) Arg-42 and Arg-176 are required for nick sensing. DNA binding reaction mixtures contained 250 fmol of nicked duplex DNA and 170, 340, 680, or 1360 fmol of the indicated ligase–adenylate (proceeding from left to right in each titration series). Ligase was omitted from a control reaction (lane [−]). The products were analyzed by native gel electrophoresis. An autoradiogram of the gel is shown.
Molecular Cell 2000 6, DOI: ( /S (00) )