1. How an RNA Ligase Discriminates RNA versus DNA Damage
Jayakrishnan Nandakumar, Stewart Shuman 
Molecular Cell 
Volume 16, Issue 2, Pages (October 2004)
DOI: /j.molcel

2. Figure 1
Sealing of a Singly Nicked Substrate by T4 Rnl2 and Requirement for RNA in the 3′-OH Strand
(A) ATP requirement. Reaction mixtures contained 1 pmol of 5′ 32P-labeled nicked RNA:DNA hybrid (• in [C]), either 100 μM ATP (+ATP) or no ATP (−ATP), and Rnl2 as specified. The extent of ligation is plotted as a function of input Rnl2. (B) Requirement for RNA in the 3′-OH strand. Reaction mixtures contained 100 μM ATP, 1 pmol of 5′ 32P-labeled nicked duplex substrate (indicated by the symbols in [C]), and Rnl2 as specified. (C) The structures of the annealed three-piece substrates are illustrated, with ribonucleotides shaded in gray and deoxyribonucleotides unshaded. The 5′ 32P-label at the nick is indicated by a dot. (D) Single-turnover ligation. Reaction mixtures contained 50 nM of 5′ 32P-labeled nicked duplex substrate with an all-RNA 3′-OH strand (■ in [F]) or a 3′-OH strand with a single 3′-deoxy substitution (○ in [F]). The distribution of ligated 42-mer product and the DNA-adenylate intermediate (AppDNA), expressed as the fraction of the total 32P-labeled material, is plotted as a function of time. (E) Substrate binding. Reaction mixtures contained 0.25 pmol of nicked substrates ■ or ○ (as shown in [F]) and increasing amounts of Rnl2 (0, 0.125, 0.25, or 0.5 pmol).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Delineating the RNA Requirement in the 3′-OH Strand
(A) Reaction mixtures containing 100 μM ATP, 0.5 pmol of 5′ 32P-labeled nicked duplex substrate (indicated by the symbols in [B]), and Rnl2 as specified were incubated for 10 min at 22°C.
(B) The structures of the annealed three-piece substrates are illustrated, with ribonucleotides shaded in gray. All other positions are deoxyribonucleotides. The 5′ 32P-label at the nick is indicated by a dot.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
A Deoxyribonucleotide at the Penultimate Position of the 3′-OH Strand Slows Ligation and Inhibits Nick Recognition
(A) Single-turnover ligation reaction mixtures contained 50 nM of 5′ 32P-labeled nicked duplex DNA substrate with 2 ribonucleotides (□) or 1 ribonucleotide (○) at the 3′-OH side of the nick. The distribution of ligated 24-mer product and the AppDNA intermediate, expressed as the fraction of the total 32P-labeled material, is plotted as a function of time. The structures of the substrates are shown below the graph; ribonucleotides are in shaded boxes.
(B) Substrate binding reaction mixtures contained 0.25 pmol of 5′ 32P-labeled nicked duplex DNA substrate with 2 or 1 ribonucleotides at the 3′-OH side of the nick and increasing amounts of Rnl2 (0, 0.125, 0.25, or 0.5 pmol).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Effects of 2′ Sugar Modifications of the Penultimate Nucleotide of the 3′-OH Strand on Steady-State Ligase Activity
The structures of the annealed three-piece substrates are illustrated, with the 3′ ribonucleotide shaded in gray and the flanking cytidine nucleotide containing different 2′-sugar substituents indicated by a question mark. All other positions are deoxyribonucleotides. Ligation reaction mixtures contained 50 mM Tris-acetate (pH 6.5), 40 mM NaCl, 5 mM DTT, 1 mM MgCl2, 100 μM ATP, 0.5 pmol of 5′ 32P-labeled nicked duplex substrate, and Rnl2 as specified (A, B, D, E, and F) or 50 mM Tris-HCl (pH 7.5), 40 mM NaCl, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, and Chlorella virus DNA ligase as specified (C). The extents of ligation of the 2′-OH and 2′-OCH3 substrates are plotted as a function of input Rnl2 in (A) and (B), respectively. The extents of formation of the ligated product and the AppDNA intermediate are plotted in (D), (E), and (F) for the 2′-H, 2′-NH2, and 2′-F substrates, respectively.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Effects of 2′ Sugar Modifications of the Penultimate Nucleotide of the 3′-OH Strand on Single-Turnover Ligation and Nick Binding
(A) Single-turnover ligation reaction mixtures contained 50 nM of 5′ 32P-labeled 24 bp nicked duplex DNA substrate (see Figure 4) with a 2′-OCH3, 2′-NH2, or 2′-F nucleoside sugar at the penultimate position on the 3′-OH side of the nick. The distribution of ligated 24-mer product and the AppDNA intermediate, expressed as the fraction of the total 32P-labeled material, is plotted as a function of time.
(B) Substrate binding reaction mixtures contained 0.25 pmol of 5′ 32P-labeled 24 bp nicked duplex DNA substrate with the indicated sugar modification and increasing amounts of Rnl2 (0.125, 0.25, or 0.5 pmol, from left to right in each titration series). The mixtures were analyzed by native PAGE. An autoradiogram of the gel is shown. Rnl2 was omitted from control reaction mixtures shown in lanes marked with a dash.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Effects of a 2′-OCH3 Modification of the Terminal Nucleotide of the 3′-OH Strand
(A) Steady-state ligation reaction mixtures contained 100 μM ATP, 0.5 pmol of 5′ 32P-labeled nicked duplex substrate with either a 2′-OH, 2′-OCH3, or 2′-H sugar substituent at the nick and Rnl2 as specified.
(B and C) Single-turnover ligation reaction mixtures contained 50 nM of 5′ 32P-labeled 24 bp nicked duplex DNA substrate with a 2′-OCH3 (B) or 2′-H (C) nucleoside sugar at the nick. The distribution of ligated 24-mer product and the AppDNA intermediate, expressed as the fraction of the total 32P-labeled material, is plotted as a function of time.
(D) The structures of the reactive termini of the substrates are depicted schematically. The template strands are not shown.
(E) Substrate binding mixtures contained 0.25 pmol of 5′ 32P-labeled 24 bp nicked duplex DNA substrate with the indicated sugar modification and increasing amounts of Rnl2 (0, 0.125, 0.25, or 0.5 pmol).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Structural Features of Rnl2 Required for Nicked Duplex Binding
Binding reaction mixtures (10 μl) containing 50 mM Tris-acetate (pH 6.5), 40 mM NaCl, 5 mM DTT, 5% glycerol, 0.25 pmol of the indicated 5′ 32P-labeled nicked duplex DNA substrate containing 3 ribonucleotides at the 3′-OH side of the nick, and increasing amount of wild-type (WT) Rnl2, the N-terminal adenylyltransferase/ligase domain Rnl2-(1-249), the K54A mutant of full-length Rnl2, or the K35A mutant of full-length Rnl2 (0.125, 0.25, or 0.5 pmol, from left to right in each titration series) were incubated for 10 min at 22°C. The mixtures were analyzed by native PAGE. Rnl2 was omitted from control reaction mixtures shown in lanes marked with a dash.
Molecular Cell  , DOI: ( /j.molcel )