Polynucleotide 3'-terminal phosphate modifications by RNA and DNA ligases

Abstract

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3'-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3'p substrate to generate an RNA 2',3'-cyclic phosphate or convert DNA3'p to ssDNA(3')pp(5')A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3'p. These modifications of RNA and DNA 3'-phosphates are similar to the activities of RtcA, an RNA 3'-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3'p or DNA 3'p to generate the adenylated intermediate. For RNA (3')pp(5')A, the third step involves attack of the adjacent 2' hydroxyl to generate the RNA 2',3'-cyclic phosphate. These steps are analogous to those in classical 5' phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3'p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3'-phosphorylated nicks in double-stranded DNA to produce a 3'-adenylated product. These 3'-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5'Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3'-terminal phosphates.

Keywords: DNA Enzyme; Enzyme Mechanism; RNA; RNA 2′,3′-Cyclic Phosphate; RNA Ligase; RNA Metabolism; Thermophile.

FIGURE 1.

RNA 3′-terminal phosphate modifications with MthRnl. A, gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p (column I) or FAM-RNA17(OMe)p (column II) followed by phosphatase treatment with either AnP or PNK. Lanes 1, 7, 8, and 14 (input) were control reaction mixtures without enzymes. Additional controls included incubation with only AnP (lanes 2 and 9) or only PNK (lanes 3 and 10). The reactions treated with MthRnl (lanes 4–6 and 11–13) were further treated with either AnP (lanes 5 and 12) or with T4 PNK (lanes 6 and 13). The reaction conditions and buffers are described under “Experimental Procedures.” The products were analyzed on urea-PAGE and visualized with SYBR Gold staining. Positions of oligonucleotide markers are indicated on the left. B, fragment analysis (capillary electrophoresis) of 3′-phosphate modifications produced by various ligases (1^st enzyme, right panel) of either FAM-RNA17p (column I) or FAM-RNA17(OMe)p (column II), followed by phosphatase treatment with either AnP or PNK (2^nd enzyme, right panel). A dashed line indicates no enzyme treatment. The reactions described in A are presented in panels 1–12. As a positive control, 10 pmol of FAM-RNA17p or FAM-RNA17(OMe)p were treated with RtcA (panels 13 and 16). These products were additionally treated with AnP (panels 14 and 17) or T4 PNK (panels 15 and 18). The T4 RNA ligase 1 and 2 reactions with FAM-RNA17p or FAM-RNA17(OMe)p were incubated at 25 °C for 2 h in the buffer supplied by the manufacturer (panels 19–26). Some reactions were also treated with AnP (panels 20, 22, 24, and 26). Substrates and corresponding products are indicated at the bottom and are color-coded. Substrates are shown in red, the products of primary enzymes and unreactive substrates of secondary enzymes are shown in green, and the products of secondary enzymes are shown in blue. The DNA standards are not colored. C, schematic of the enzymatic reactions in A and B.

FIGURE 2.

A single-stranded DNA 3′-terminal phosphate modification by thermophilic RNA ligases. Enzymes and reactions for each experiment are indicated at the tops of the panels. A, gel shift analysis of reaction products of DNA17p with MthRnl, TVa, and TS2126. Reaction mixtures were incubated at 65 °C for 60 min, heat-inactivated at 90 °C for 5 min, and treated with 10 μg of proteinase K at 37 °C for 60 min. The products were analyzed on 15% urea-PAGE and visualized with SYBR Gold staining. Positions of a substrate (ssDNA17p) and an adenylated product (ssDNA17ppA) are indicated on the right. Positions of oligonucleotide markers are indicated on the left. C, control. B, pH dependence of α-[³²P]AMP incorporation into DNA17p using 5 pmol of a DNA17p substrate, 10 pmol of MthRnl under the conditions described for A (except for the indicated variable pH), and supplement with 1 μCi of α-[³²P]ATP. B I, reaction products were treated and separated as described for A. B II, radioactive products visualized by PhospoScreen scanning. Mr is the oligonucleotide molecular weight marker. C, comparison of 5′- and 3′-phosphorylated ssDNA adenylation with MthRnl. Reactions were performed as described in A using 5 pmol of pDNA17-NH₂ or DNA17p and variable amounts (5.000–0.625 pmol) of enzyme. The molar ratio of substrate to enzyme (S/E) is listed at the bottom of the gel. D, comparison of 5′- and 3′-phosphorylated ssDNA adenylation with RtcA. Reactions were performed and analyzed as described for MthRnl in C but at 37 °C with the variable substrate-to-enzyme ratios indicated at the bottom. E, deadenylation of 3′- and 5′-phosphate-modified ssDNA with yeast 5′Deadenylase. 5 pmol of DNA17ppA and AppDNA17-NH₂, the products of MthRnl modification described in legend for A, were treated with serially diluted 5′Deadenylase at 25 °C for 30 min. The products were analyzed by gel electrophoresis as described in A. Variable substrate-to-enzyme ratios are indicated at the bottom. F, K97A MthRnl mutant activity assays using 5 pmol ssDNA17p substrate were performed and analyzed as described for wild-type enzyme in C.

FIGURE 3.

Modification of the 3′-phosphorylated nick in duplex DNA by various ligases. A, structure of the double-stranded substrate used in an assay and 3′-adenylation reaction. B, control for double-stranded DNA formation. After annealing of three oligonucleotides as described under “Experimental Procedures,” a 5-pmol aliquot was digested with the restriction endonuclease BanI for 90 min at 25 °C, heat-treated, and analyzed on 15% urea-PAGE. FAM-labeled oligonucleotides were visualized by fluorescent scanning. Lane 1 (input) is a reaction mixture without enzyme, and lane 2 is the product of restriction enzyme digestion. The positions of the FAM-DNA17p substrate and six-nucleotide-long FAM-DNA6 product are indicated on the right. C, gel shift analysis of FAM-labeled reaction products of the 3′-phosphorylated nick of duplex DNA substrate using 15% urea-PAGE and fluorescent scanning. Reactions with T4, T3, T7, PBCV1 DNA ligases (lanes 2–5), and RtcA (lane 6) were performed for 4 h and with MthRnl, TVa, and TS2126 archaeal RNA ligases (lanes 7–9) for 20 h at 25 °C. Lane 1 (input) is a reaction mixture without enzyme. The positions of the FAM-DNA17p oligonucleotide of the double-stranded substrate and its FAM-DNA17ppA product are indicated on the right, and enzymes used in the assay are shown at the bottom. D, reactions of 3′-phosphorylated single-stranded DNA with DNA ligases were performed under the same conditions as those described for the nicked dsDNA substrate in C. Lane 1 is a positive control reaction of ssDNA17p with MthRnl. Lane 2 (input) is a reaction mixture without enzyme. Lanes 3–6 are reactions with T4, T3, T7, and PBCV1 DNA ligases, respectively. The positions of the ssDNA17p substrate and ssDNA17ppA product are indicated on the right, and enzymes used in the assay are shown at the bottom.