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Yeast Ribonuclease H(70) Cleaves RNA-DNA Junctions

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Yeast Ribonuclease H(70) Cleaves RNA-DNA Junctions View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 206, number 2 FEBS 4062 October 1986 Yeast ribonuclease H(70) cleaves RNA-DNA junctions Robert Karwan and Ulrike Wintersberger Department of Molecular GEnetics, Institute for Tumorbiology and Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Wien, Austria Received 7 August 1986 A specific substrate, Ml3 DNA:RNA-[3zP]DNA, was synthesized to investigate the mode of cleavage of enzymes with RNase H activity. RNase H(70) from Saccharomyces cerevisiae hydrolyzes the phosphodiester bond at the RNA-DNA junction of this substrate, thereby producing a 5’-monophosphate-terminated poly- deoxyribonucleotide and 3’-hydroxyl-terminated oligoribonucleotides. RNase H DNA-RNA hybrid DNA replication RNA primer removal (Saccharomyces cerevisiae) 1. INTRODUCTION ing short RNA primers. The phosphorus atom of the phosphate bridging the RNA and DNA parts Recently we have purified a ribonuclease H of this polynucleotide is radiolabeled (see fig.1). (RNase H, i.e. an enzyme which specifically Here we show that, in vitro the yeast RNase H(70) hydrolyzes the RNA strand of a DNA-RNA preferentially hydrolyzes the phosphodiester bond hybrid) from the yeast, Saccharomyces cerevisiae, between the ribonucleotide and the deox- which stimulates the in vitro DNA synthesis by yribonucleotide at the RNA-DNA junction. DNA polymerase A from the same organism [ 1,2]. Under certain conditions this protein also exhibits 2. MATERIALS AND METHODS reverse transcriptase activity [3]. As with RNases H from other organisms [4] no definite RNase H(70) and DNA polymerase A from S. physiological role could be attributed to the en- cerevisiue as well as M13mp9 DNA were purified zyme until now. DNA-RNA hybrids occur during as described [1,6,7]. DNA polymerase I (Klenow cellular DNA replication [5], both during the in- fragment) and RNA polymerase from E. cofi were itiation of leading strand synthesis as well as dur- from Boehringer/Mannheim. ing the discontinuous polymerization of the lagging strand whereby each nascent Okazaki frag- 2.1. Preparation of the Ml3 DNA : RNA[P*JDNA ment contains a short RNA primer covalently at- substrate tached to the 5 ‘-end of the DNA piece. A plausible Single-stranded M13mp9 DNA was incubated role for an RNase H would be its responsibility for for 10 min with all 4 ribonucleoside triphosphates the removal of these RNA primers. To determine in presence of MnC12 (4 mM) to favor nonspecific whether the enzyme isolated by us [designated transcription initiation (for detailed reaction con- yeast RNase H(70)] is, at least in vitro, capable of ditions see [ 11). The reaction was stopped by addi- such a task we have tested it with a substrate tion of EDTA (50 mM). The hybrids were purified specifically constructed for this purpose. The by 3 successive spun columns [7], phenol extrac- substrate (which we term Ml3 DNA: tion and ethanol precipitation. Then they were RNA[P*]DNA) consists of a closed circle of Ml3 reacted for 2 min at 30°C with Klenow polymerase DNA with complementary DNA strands contain- (0.1 pmoV0.6 pmol Ml3 DNA) and 5 PM Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50 0 1986 Federation of European Biochemical Societies 189 Volume 206, number 2 FEBS LETTERS October 1986 [cu-32P]dATP (5 Ci/mmol) [7]. The reaction products were purified as above and further elongated by incubation with 25 units/ml of yeast *$T DNA polymerase A [6] and all 4 unlabeled deox- mode of yribonucleoside triphosphates for 30 min at 30°C. cleavaqe: (b)nocleavage (OHS (OHS no (OH)- 2.2. Nearest-neighbour analysis radioactlve M 13 DNA : RNA[P*]DNA (10 pM ribonucleo- products: 5’-P*-polydeoxyribo- 2CS’)NMPk 3’-P*-oligoribo- tides/l50,J assay mixture) was incubated with nucleotides (a$.) (a.s.1 nucleotides(a.s.) 0.1 pg RNase H(70) at 30°C and aliquots (20 ~1) Fig.]. Scheme of experiments to analyze the mode of were removed after different time intervals. These cleavage of an RNA-DNA junction by an RNase H (see were made 50 mM in EDTA and trichloroacetic text for explanation; a.p., acid-precipitable; a.s., acid- acid was added to a final concentration of 10%. soluble). The complementary DNA strand of the After centrifugation the precipitates were substrate is omitted in the drawing. repeatedly solubilized in 0.5 M KOH and precipitated in 5% trichloroacetic acid; finally (3 ‘-[P*]oligoribonucleotides which can be dis- 10~1 aliquots were hydrolyzed with 0.3 M KOH criminated from mononucleotides chromato- for 20 h at 37°C. The remaining polydeoxyribo- graphically). Thus, by reacting Ml3 DNA : nucleotides were again precipitated (5% trichloro- RNA[P*]DNA with RNase H(70) and alkali, and acetic acid), washed and analyzed by electro- by analyzing the reaction products one can phoresis in denaturing agarose gels (0.7%) [8]. Size distinguish between all possible modes of cleavage. standards were Hind111 fragments of ADNA [7]; The results of our tests are depicted in figs 2 and autoradiography was carried out for 48 h [7]. The 3. Incubation of Ml3 DNA : RNA[P*]DNA with supernatants from the first trichloroacetic acid RNase H(70) alone (reaction a of fig.1) does not precipitations were lyophilized, solubilized in noticeably change the size distribution of the acid- water, neutralized and analyzed by ascending thin- precipitable polymers (cf. lanes 1 and 2 of the layer chromatography on polyethyleneimine-cel- denaturing gel electrophoresis in fig.2). This means lulose in 1 M acetic acid/3 M LiCl (9: 1, v/v). that the substrate contains DNA chains of between 2 and 4 kb primed by short oligoribonucleotides, hence reflecting the in vivo situation during lagging 3. RESULTS strand DNA replication. It may be interesting to The rationale of the nearest-neighbour analysis note that yeast DNA polymerase A which was used for testing the specificity of RNase H(70) is for the RNA primer elongation reaction must have described in fig. 1. If Ml3 DNA: RNA[P*]DNA behaved quite processively during the polymerisa- (see sections 1 and 2) is reacted with a nuclease able tion process. No radioactivity is made acid soluble to hydrolyze the bond between the last during incubation of the substrate with RNase ribonucleotide and the labeled 5 ’ -phosphorus of H(70) alone (lane 2 of fig.3) indicating that RNase the first deoxyribonucleotide the label will stay H(70) cannot have cleaved the ester bond between with the acid-precipitable reaction products. No the labeled phosphate and the 5 ‘-end of the DNA acid-soluble radioactive ribonucleoside mono- part of the substrate (a reaction which would cor- phosphates should be detectable after subsequent respond to proposal c of fig.1). The acid-soluble alkaline hydrolysis (reaction a + (OH)- in fig.1). products of the reaction of Ml3 By contrast, alkaline hydrolysis alone (experimen- DNA: RNA[P*]DNA with alkali alone (reaction b tally or, because the RNase H cannot cleave a junc- of fig.1) consist, as expected, of 2’(3’)- tion between RNA and DNA, expt b in fig.1) will ribonucleoside monophosphates (fig.3, lane 3) and result in labeled ribonucleoside monophosphates. no radioactivity is left to the DNA part of the Cleavage of the bond between the phosphorus and substrate (fig.2, lane 3). If the substrate was in- the 5’-end of the DNA part at the RNA-DNA cubated with RNase H(70) for increasing periods junction (reaction c in fig. 1) would lead to labeled of time before alkaline hydrolysis the radioactivity products only in the acid-soluble fraction was increasingly found with the acid-precipitable 190 Volume 206, number 2 FEBS LETTERS October 1986 Treatment: 1 234567 fig.1. An identical experiment using the same substrate, but labeled throughout the RNA primer part, was carried out in parallel. The amounts of RNA counts rendered soluble by RNase H(70) alone were 2, 7, 16 and 30% after 1, 2.5, 5 and 10 min incubation, respectively (not shown). As complete cleavage of the RNA-DNA junction is obtained after 10 min this reaction obviously takes a faster course than total RNA hydrolysis. 4. DISCUSSION Our data show that RNase H(70) from yeast is able to hydrolyze the phosphodiester bond be- tween the 3 ’ -oxygen and the phosphorus preferen- 0.6- tially at a RNA-DNA junction. The products of Fig.2. Denaturing agarose gel electrophoresis of acid- this reaction are a 3 ’ -OH-terminated precipitable products from the reaction of Ml3 oligoribonucleotide and a 5 ’ -monophosphate- DNA: RNA[P*]DNA with RNase H(70) followed by terminated polydeoxyribonucleotide, the latter be- treatment with alkali. Numbers on the left: size markers ing a substrate for DNA ligase. Thus, RNAse in kb. H(70) is suited for and might well be involved in primer removal during in vivo DNA replication. In yeast the situation is different from E. coli Treatment: 1 2 3 4 5 6 because, in contrast to E. coli DNA polymerase I, RNaseH(7O)(min) [ 0 1 l0 1 0 2.51 5 there is no yeast DNA polymerase exhibiting a Alkali 3 ’ -5 ’ -exonuclease activity [9] which could be used for primer degradation. Therefore, a separate pro- 2’(3’)AM P - tein must be assumed for this function in yeast and possibly in other eukaryotes. Interestingly, yeast RNase H(70) resembles more closely the proteins from retroviruses which exhibit RNase H activity [3] than the RNase H from E. coli [4,10]. The S’ATP - RNase H activity, e.g. of AMV reverse transcrip- tase, does remove RNA primers and is able to hydrolyze precisely and preferentially the junction Start - between RNA and DNA at the origin for plus- strand synthesis of proviral DNA [l 11.
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