DNA polymerization catalysed by a group II intron RNA in vitro
DNA polymerization catalysed by a group II intron RNA in vitro Martin Hetzer, Rudolf J. Schweyen and Manfred W. Mueller*
Vienna Biocenter, Institute of Microbiology and Genetics, University of Vienna, Dr Bohr-Gasse 9, A-1030 Vienna, Austria
Received December 4, 1996;Revised and Accepted March 10, 1997
ABSTRACT
The excised group II intron bI1 from Saccharomycescerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro. Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3' -> 5' direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3' -> 5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.
INTRODUCTION
The discovery of RNAs with true enzymatic activities (ribozymes) (1 ,2 ) has provoked interest in exploring the catalytic potential of RNA. Meanwhile, various chemical reactions have been shown to be catalysed by members of the group I and group II intron ribozymes with substrates as different as RNA, DNA and even aminoacylated derivatives of RNA (3 -13 ). These results have repeatedly been discussed in the light of the role of RNA in a presumed RNA world (10 ,12 ,14 -19 ). Special interest has focused on RNA-catalysed polymerization activities of some introns, since such reactions might have been involved in the development of the first self-replicating systems (20 ).
Group II introns excise autocatalytically from pre-mRNAs through two separate transesterification reactions in vitro (21 -24 ). In the first step, the 2'-OH group of the intron internal branch point adenosine attacks the 5' splice site (5' ss) to produce 5' exon and intron-3' exon intermediate RNA in lariat form. In the second step the 5' exon attacks the 3' ss resulting in exon ligation and release of the lariat intron RNA.
The released lariat RNA can catalyse the reversal of exon ligation (9 ) with different substrates, either composed of RNA, DNA or hybrid molecules containing both types of nucleic acids (11 ,12 ,25 ; Fig. 1 A). Substrate specificity in reverse splicing is provided by intermolecular base pairing interactions (9 ,11 ). A sequence motif of 6 nt in the 5' exon substrate RNA (IBS1, for intron binding site 1) that is complementary to an intron internal motif (EBS1, for exon binding site 1) (26 ) is important to activate the phosphorous atom 3' adjacent to the IBS1 motif for nucleophilic attack by the 3'-terminal hydroxyl group of the lariat- IVS (intervening sequence) RNA (11 ). This transesterification reaction represents reversal of the exon ligation reaction and leads to a charged lariat (Fig. 1 A). The 3' exon sequences can be functionally substituted by deoxyribonucleotides, mononucleotides and even single phosphate groups (11 ). The charged lariat itself can be discharged (i.e. exon ligation) and the 3' exon part is transferred to the 5' exon (or a 5' exon substitute), leading either to ligated exons or to recombined RNA/DNA molecules (11 ,12 ; Fig. 1 A). On the basis of alternating charging and discharging reactions, the catalytic group II ribozyme repertoire exhibits different activities, such as a 3'-terminal transferase, RNA-DNA and DNA-RNA ligase, monophosphotransferase and general recombinase (11 ,12 ).
MATERIALS AND METHODS
RNA synthesis
Run-off transcripts from bI1 were synthesized in vitro with T3 RNA polymerase in the presence of [[alpha]-35S]UTP from EcoRI- digested plasmids Bluescript and bI1[Delta]24 as described (29 ). Pre-RNAs were gel purified and incubated in 40 mM Tris-HCl, pH 7.2, 60 mM MgCl2, 2 mM spermidine and 660 mM NH4Cl at 45oC for 60 min. The splicing product bI1 lariat-IVS RNA was quantitatively recovered from preparative polyacrylamide gels.
Synthetic oligoribonucleotides were synthesized on an Applied Biosystems DNA/RNA Synthesizer (392). 5'-End-labelling and purification were performed as described (11 ). Oligonucleotides: IBS1dA, 5'-AAAGGAAGACAGdA-3'; IBS1dG, 5'-AAAGGAAGACAGdG-3'; IBS1dT, 5'-AAAGGAAGACAGdT-3'; IBS1dC, 5'-AAAGGAAGACAGdC-3'; IBS1dGpA8, 5'-AAAGGAAGACAGdGAAAAAAAA-3'.
DNA polymerization and insertion reactions
For DNA polymerization 5' terminally [[gamma]-32P]ATP-labelled synthetic 5' exon substrates IBS1dN (5 [mu]M) labelled at the 5'-end and lariat-IVS bI1 (0.5 [mu]M) were incubated in 40 mM Tris-HCl, pH 7.2, 60 mM MgCl2, 2 mM spermidine and 1.25 M NH4Cl at 45oC. Samples were taken for different times as indicated, analysed and quantified (PhosphorImager; Molecular Dynamics).
For DNA insertion synthetic 5' exon substrates (IBS1dG) labelled at the 5'-end with [[gamma]-32P]ATP and lariat-IVS bI1 were co-incubated as described above with substrate IBS1dGpA8 (1 [mu]M). The products of the deoxyribonucleotide integration reaction were gel purified and subjected to alkaline hydrolysis or analysed by dideoxy sequencing.
Alkaline hydrolysis
Reaction products containing 5'-end-labels were incubated in hydrolysis buffer (0.5 M sodium bicarbonate/carbonate, pH 9.2) for 15 min at 90oC and analysed on denaturing 20% polyacrylamide gels.
Dideoxy sequencing of polymerization product
*pN7GACAGpdG2dG1A8 22mer (cf. Fig 3 A) was gel extracted and tagged by virtue of the 3'-terminal OH group to the 5' phosphate group of an EcoRI site-containing a DNA adapter molecule in the presence of T4 RNA ligase (38 ). The 3'-OH group of an adapter-specific primer was used to initiate cDNA synthesis, followed by CRTC (controlled ribonucleotide tailing of cDNA ends) (39 ) and covalent ligation of a KpnI site-containing DNA adapter to the 3'-OH group of the elongated cDNA product in the presence of T4 DNA ligase. PCR amplification was performed with adapter-specific primers. Dideoxy sequencing after a cloning step, utilizing restriction sites derived from the DNA adapters (KpnI and EcoRI), was performed on an automated DNA sequencer (LI-COR L4000; VBC Sequencing Service) as described (39 ).
RESULTS AND DISCUSSION
In a first step towards group II intron-catalysed DNA polymerization, we synthesized oligoribonucleotides (13mers) containing single deoxyribonucleotide substitutions (IBS1dN; dN = dA, dG, dC or dT) at the 3'-terminal IBS1 motif (*pN7GACAGdN). The four different 5' exon substrates were assayed in the presence of gel-purified bI1 lariat RNA under splicing conditions at 45oC (Fig. 2 A). All four 32P-5'-end-labelled input molecules (*pN7GACAGpdN) were progressively converted into products 1 nt shorter at the 3'-end [12mer (-1), *pN7GACAG], indicating that all four deoxyribonucleotides were accepted for the (-1) charging reaction. Additionally, products extended by one or more deoxyribonucleotides in length were observed in all four assays [e.g. 14mer (+1), *pN7GACAGdA2dA1].
