Copyright 0 1995 by the Genetics Society of America
Short-Patch Reverse Transcription in Escherichia coli David S . Thaler, Gregory Tombline and Kenneth Zahn Laboratory of Molecular Genetics and Informatics, Rockefeller University, New York, New York 10021 Manuscript received November 9, 1994 Accepted for publication March 22, 1995 ABSTRACT Chimeras of RNA and DNA have distinctive physical and biological properties. Chimeric oligonucleotides that contained one, two or three ribonucleotides whose phosphodiester backbone was covalently continuous with DNA weresynthesized. Sitedirected mutagenesis was used to assess genetic information transfer from the ribonucleotide positions. Transfer was scored by the formation or reversion of an ochre site that also corresponded to a restriction cleavage site. This allowed physical as well as genetic assay of mutational events. Bases attached to the ribonucleotides were able to accurately direct the synthesis of progeny DNA. The results suggest that in vivo DNA polymerases utilize a "running start" on a DNA backbone to continue across a covalent backbone junction into a region of ribonucleotides and then back again onto a normal DNA backbone. The phenomenon is designated short-patch reverse transcription (SPRT) by analogy to short-patch mismatch correction and reverse transcription as the term is generally used. The possibility is considered that SPRT contributes to an unrecognized pathway of mutagenesis.
R
NA and DNA may be chemically associated in two different ways, as RNA-DNA hybrids and as RNADNA chimeras. Each association has specificphysiochemical properties (SALAZAR et al. 1993). InRNA-DNA hybrids, complementary strands of RNA and DNA are paired by hydrogen bonds. In contrast, RNA-DNA chimeras contain both ribonucleotides and deoxyribonucleotides on a covalently continuous phosphodiester backbone. Patches of ribonucleotides may be embedded within the DNA sequence of chimeras. Both hybrids and chimeras are found in biological contexts: as primers for DNA lagging-strand synthesis (OGAWA and 01980) and during the priming of replication origins, as essential intermediates in the lifecycleof retroviruses (GILBOAet al. 1979), as the result ofDNA polymerases incorporating ribonucleotides (KORNBERG and BAKER1992), andas RNA in mitochondrial chromosomes isolated from mouse cell lines (GROSSMAN et al. 1973) but not in the same DNAsequences isolated from Escherichia coli (BROWNet al. 1976). Short RNA-DNA hybrids are formed during conventional transcription (YAGER and VON HIPPEL1987; SURR A et ~al. 1991). However, the utilization of these transcripts as primers for DNA synthesis in cells deficient in RNase H mayyield longer chimeras and hybrids (MAGEE et al. 1992). This work considers the genetic properties of ribonucleotide chimeras and hybrids.Of particular interest was whether the bases attached to ribonucleotide backCorresponding authw: David S. Thaler, Laboratory of Molecular Genetics and Informatics, Rockefeller University, 1230 York Ave., New York, NY 10021. E-mail:
[email protected] Genetics 1 4 0 909-915 ( p y , 1995)
bone positions in chimeras are able to code properly. The results of this study show that ribonucleotides in DNA code accurately. This represents a new context for reverse transcription in vivo. MATERIALSANDMETHODS Oligonucleotides were synthesized by the Rockefeller University Protein Synthesis Facility.The positions of ribonucleotides were confirmed by 5"end labeling with '*P followed by alkali hydrolysis or RNase treatment, acrylamide gel electrophoresis, and autoradiography (data not shown). In vitro annealing and DNA synthesis were camed out in standard ways (SMITH1985). Primers were phosphorylated by adding 50 pmol of primer to 20 pl of 50 mM Tris-HC1 (pH 7.6), 10 mMMgC12, 5 mM dithioerythritol (DTE), 0.1pM spermidine HC1, 0.5 mM ATP, and 5 units of T4 polynucleotide kinase. After incubating at 37" for 1 hr, the reactions were inactivated by heating to 70" for 10 min. Approximately 5 pmol (2 pl) wereremoved and directly added to0.4 pg (-0.16 pmol) of singlestranded uracil-containing phagescript DNA prepared by themethod of KUNKEL (1985). This primer/template mixture was brought to a final volume of 10 pl in 20 mMTris-HC1 (pH 7.5), 2 mMMgCl?, and 50 mMNaCl. Mineral oil (30 pl) was then overlaid to prevent evaporation. The reaction tube was heated to 90" and cooled slowly to room temperature. Synthesis buffer (7 p1) was then added such that the final 20-pl reaction volume contained 25 mMTris-HC1 (pH 7.5), 5 mMMgC12,25mMNaCl, 1.5 mM DTE,500pM each dNTP, and 0.7 mMATP. T4 gene 32 protein (0.75 pg) was added, and the reactions were kept at room temperature for 10 min, after which 4 units of T4 DNA polymerase were added and the reactions were maintained at 0" for 5 min and then raised to room temperature for 15 min. T4 DNA ligase (8 units) was added, followed by incubation at 37" for 4-5 hr. Reactions were terminated by adding 80 pl of sterile distilled water [treated with 0.1% diethylpyrocarbonate (DEPC) before autoclaving] and were immediately extracted with equal volumes of pheno1:chloroform:isoamyl alcohol (2524:1) followed by chloroformisoamyl alcohol
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D. S. Thaler, G. Tombline and
(241). Synthesis products were precipitated in 100 mM sodium acetate (pH 5.3), 67% ethanol at -20" overnight followed by centrifugation. The pellet was washed with70% ethanol,centrifugedagain,anddriedunder vacuum with centrifugation.Pelletswereresuspendedin 100 p1ofTE buffer (treatedwith 0.1% DEPC before autoclaving). Synthesis and ligation were assessed on 1% agarose gels buffered with Tris-acetate EDTA(TAE) run at 10 V/cm for 1 hr and then stained in 0.5 pg/ml of ethidium bromide. Aliquots(typically 3 pl into 40 p1 of electrocompetent cells prepared according to the protocol suppliedby Stratagene)were electroporated into NM522 (F' lac 19 lacZAMI5 poA+B+/supE thi A (lac-proAB) A ( hsdMSmmB) 5 rk- mk- McrB-) (GOUGH and MURRAY1983), whichsupplies lac0 complementation, and plated on E medium containing thiamine, 0.2% glucose, 6 pg/ml IPTG and 2 pg/ml5-brom&hloro-3-indolyl-~-D-galactopyranoside (X-Gal, Diagnostic Chemicals Limited). Blue and white plaques were counted after overnight incubation at 37". Phage plaques were purified by streaking onto lawns of NM522 cells;the bacteriallawns were madefirm by refrigeration at4" for 230 min before use. Well-isolated plaques were individually picked with Pasteur pipettes, dispersed into 200 pl of SM buffer (ARBER et al. 1983) and stored at 4".Each isolate in SM buffer was tested for suppressibility by replicaspotting ontolawns of NM522, and NM522, containing a plasmid carrying an ochre-suppressor tRNA gene (MASSON and MILLER 1986). SM buffer were used inPCR Aliquots of the same isolates in reactions. The sequences and locations of the primers are shown in Figure 1. For PCR, 5 p1 of each isolate inSM buffer were preheated at 94" for 10 min in 100 pl of PCR reaction mix containing 7 pmol of each primer, 0.25 mM total NTPs (equal dATP, dCTP, dGTP and dTTP), 10 mM Tris-HC1 pH 8.3, 50 mM KC1 and 2.5 mM MgC12 overlaidwith30p1of mineral oil. After preheating, 0.5 plof 5 units/pl AmpliTaq DNA polymerase (Cetus) were added and PCR was carried out using a cycling program of 94" for 30 sec, 50" for 1 min and 70" for 2 min. PCR was run for 25 cycles and was followed by 15 min at 72". Five-microliter aliquotsof the PCR products were digested separately with XbuI and Hind111 in 20-pl reactions carried out in 96well microtiter plates. Electrophoretic analysis was as described above. RESULTS The experimental design is shown in Figure 1. Double-stranded DNA phage substrates containing ribonucleotides atdefined positions in one strand were constructed by in vitro DNA synthesis and then electroporated into E. coli. Oligonucleotides containing one, two or three ribonucleotides were used as primers for the in vitro DNA synthesis over a template of a singlestrandedphage fl containing the lucza! gene. Mismatches were created between the DNA of the template luc2a! geneand ribonucleotide(s)inthe synthesis primer.These ribonucleotide-DNA mismatches were assayable in two ways: the site of the mismatch defines an ochre stop codon in lacza! and the same site is also essential for restriction by XbuI. Together, the ochre marker and restriction site clearly distinguish between specific and nonspecific mutagenesis. Experiments were performed in two complementary combinations, with the template being ochre and null
J L Zahn
for the XbuI restriction site and with wild-type information present on theribonucleotide-containing oligonucleotide (Table l ) ,or, conversely, with the lac&+ XbuI' template and the ochreallele, which obviates the XbuI' site on the ribonucleotide-containing oligonucleotide (Table 2). In the first case, the template chain yields white (ochre) plaques on a nonsuppressing host; the appearance of blue plaques (with the X6uI site restored) indicates information transfer from the oligonucleotide. In the second case, white plaques are candidates forinformation transfer fromthe oligonucleotide. White plaques were tested for the suppressible ochre mutation; white ochre plaques yield blue plaques when assayed on a suppressing host. Loss of the XbuI restriction site was assessed by digestion of a PCR-amplified 2.2-kb segment of candidate phage. Cleavage of the nearby Hind111 site was used as a positive control for the fidelity ofDNA synthesis and digestibility of the DNA (Figure 2). The primary result of this study is that one-, two- or three-base stretches of ribonucleotides transfer information to progeny DNA in vivo in wild-type E. coli (Tables l and 2). When thetemplate is ochre and the oligonucleotide contains wild-type information,the mismatch is T:C withC being the ribonucleotide(Table 1; Figure 1).With a single ribonucleotide at the informative position, 0.64% of the progeny phage have become wild-type at the ochreposition asjudged by restoration of the XbuI site (Table 1, row 1). With two contiguous ribonucleotide positions, 11%of the progeny phage were lact and harbored a restored X6uI site (Table 1, row 2).With three ribonucleotide positions, the yield of XbuI phage was an intermediate 2.8% (Table l, row 3). A similar pattern, inwhich two contiguous ribonucleotides supported the most information transfer, was seen in the complementary experiment in which the template is wild-type and the mutant allele is on the oligonucleotide (Table 2). The mismatch is G:A, with A being the ribonucleotide. One ribonucleotide yielded 0.34%information transfer as scored by both ochre and XbuI conversion (Table 2, row 1).With two contiguous ribonucleotides,theproportion of converted phage yield rose 10-fold to 3.4% (Table 2, row 2). With three ribonucleotides, theproportion of convertedphage dropped to 0.02% (Table 2, row 3 ) . Importantcontrolsincluded in these experiments were asfollows: (1) an oligonucleotide that contains one, two or three ribonucleotides at the site of the marker mutation that is not mismatched with the DNA template(Table 1, rows 5 , 6, 7; Table 2, rows 5 , 6, 7) ; (2) a DNA oligonucleotide that covers the site and contains the mutation, a control that simply recapitulates standard DNA-oligonucleotidedirectedmutagenesis (SMITH1985) and allows a comparison of the efficiency and specificity ofinformation transfer mediated by RNA us. DNA at the critical position (Table 1, row
Short-Patch Reverse Transcription
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0LIGQNUCZM)TIDE PRlMERs RNA positions are
R l wild type R l ochre R2 wild type R2 ochre R3 wild type R3 ochre D wild type D ochre
T7 primer FCRprimerl Fatprimer2
GGCGAGATmGATC 5' GGCGAGATaGATC5' GGCGAGPJLLTTG?iTC5' GGCGA-GATC5' GGCGA-GATC5' GGCGA-GATC5' GGCGAGATCITGATC5' GGCGAGATATTGATC5' GGATATCACTCAGCAT5' lTI'TATKACKGTGTCTG5' GGGCGITTICATAA'IGTCC5'
FIGURE1.-The experimental design. The procedure is derived from the standard method of oligonucleotidedirected mutagenesis (SMITH1985). The oligonucleotides in this study contain ribonucleotides to assess the ability of RNA backbone positions to contribute genetic information. The 7.372-kb single-stranded template was the flderived vector phagescript (Stratagene). The sequences of oligonucleotides used in this study are given in the lower part of the figure. Ribonucleotide positions in the oligonucleotides are designated by bold underlined letters. The polylinker sequence and primer location are shownin an expanded version underneath the circle that marks them on the overall vector map.
4; Table 2, row 4); ( 3 ) a DNA oligonucleotide that covers the critical site but does not contain a mismatch (Table 1, row 8; Table 2, row 8); and (4) a DNA oligonucleotide that does not cover the ochre site but will act as a primer and support synthesis over the critical site (Table 1, row 9). Mutations in this case are the result of errors that occur during in vitro synthesis (KUNKEL and ALEXANDER 1986). DISCUSSION
These experiments show that chimeras composed of one, two or three ribonucleotides embedded in an oth-
erwise DNA backbone and used in a standard site-directed mutagenesis protocol on bacteriophage fl are genetically active. The information coded by bases attached to the ribonucleotide backbone is inherited by progeny DNA. The quantity of information transfer from the chimeras ranged from a low of 0.02% (Table 2, row 3 ) to a high of 11% (Table 1, row 2). At present, nothing can be concluded concerning the causes of these quantitative differences due (apparently) to the different ribonucleotide-DNA mismatches and their contexts; this is an area for further work. Some candidate mechanisms for sequence specificity include the following: (1) mis-
912
D. S. Thaler, G. Tombline and K Zahn TABLE 1 Conversion of ochre, Xbd- DNA by wild-type RNA
Primer
Row
R1 wild-type
1
64)
(96) 2
R2 wild-type
3
R3 wild-type D wild-type
4
.3)
(97) R1 ochre
5
.01)
(33) 6
R2 ochre 2/5
7
R3 ochre
80/10,366
D ochre 0/2
9
T7
10
None
Blue/total
65/10,144 68/10,144 (0.67) 22/209 23/209 (11) 12/427 12/427 (2.8) (2.8) 408/4,267 (9.56) 3/ 14,035 (0.02) 5/994 (0.5) 0/300 (50.3) 2/10,366 (0.02) 3/6,439 (0.05) 0/8,645 (50.01)
Xbu+/blue
(Blue/XbuI+)/total
65/68 22/22 (100) 12/12 (100) 68/70 113
(40) NA
(11)
[408/4267] * [68/70] 1 / 14,035
2/994 (0.2) 0/300 (50.3)
NA 0/3 NA NA
(50.01) 0/6,439 (50.02) 0/8,645 (50.01)
Values in parentheses are percentages.
match correction, which is known to respond in a quantitatively distinct manner depending on both thebasebase mismatch and its sequence context (KRAMER et al. 1984; DOHET et al. 1985; JONES et al. 1987; MODRICH 1991); (2) the ung restriction system, which is known to act on uracil residues in a DNA backbone and may have relevant enzymatic activity on ribonucleotides in a DNA context (HATAHET et al. 1994), although this is rendered less likely by recent structural studies (SAWA et al. 1995); and (3) possible sequence constraints on the in vivo abilities of DNA polymerases to utilize a running start on a DNA backbone to continue across a covalent backbone junction into region a of ribonucleotides and then back again onto a normal DNA backbone. Only limited conclusions can be drawn concerning the possibilityof nonspecific mutagenesis associated with ribonucleotides in the template. If random misincorporation opposite ribonucleotides were the major mechanism, then the sequence of the RNA oligonucleotide should not affect the reversion rate. Sequence specificity in both orientations demonstrates that the ribonucleotides can act as faithful templates. The possibility remains open that ribonucleotides may sometimes act as unfaithful templates or otherwise promote mutagenesis at their site of incorporation. (See Table 1, rows 5 and 6 for a trace of evidence in favor of random mutagenesis.) No polymerase is completely accurate, and the backbone junction between DNA-RNA could well be a site for special activities. Even if ribonucleotide-containing chimera templates support highly accurate DNA polymerization, ribonu-
cleotide incorporation into DNA is a possible route to mutagenesis in vivo if the ribonucleotide incorporation itself is error-prone. Factors that contributeto the incorporation of nonstandard nucleotide backbones may also allow noncanonical base pairs. Manganese and nucleotide pool imbalance predispose DNA polymerases to incorporate ribonucleotides (IDEet al. 1993). These conditions are also associated with a high proportion ofbase mismatches in deoxyribonucleotide addition (LOEBand ZAKoUR 1980; TABOR and RICHARDSON 1989; MULLENet al. 1990). Therefore, a high proportion of the ribonucleotides incorporated by DNA polymerases may be mismatched against a DNA template. The error rate of transcription is reported to be 10-2-10-5 per base (BLANKet al. 1986; LIBBYand GALLANT1994), much higher thanfor normal DNA replication (ECHOLS and GOODMAN 1991). Ribonucleotide incorporation is a potential target for physiological modulation (ERIE et al. 1993). The mode of action of proofreading and mismatch correction on RNA-DNA mismatches is unknown and of interest in this context. Pol I is not more error prone during reverse transcription than during DNA replication (RICCHETTI and BUC 1993). HIV reverse transcriptase may actually be less errorprone when using an RNA than a DNA template (BOYERet al. 1992;J1and LOEB1992). However, we favor the particular hypothesis that template junctions between DNA and RNA are predisposed to misincorporation as well as other classes of genetic change. The asymmetry ofthe replication fork affords several possibilities for differential mutagenesis (KUNKEL 1992). In light of this report, incorporation of Okazaki
Short-Patch Reverse Transcription
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TABLE 2 Conversion of wild-type DNA by ochre, XbuI- RNA
blue Row
Light Primer R1 ochre R2 ochre R3 ochre
D ochre R1 wild-type R2 wild-type R3 wild-type
D wild-type None
total
White plaques/ total
plaques/white tested white
42/6,483 (0.65) 372/7,246 (5.1) 26/9,748 (0.27) 864/2,510 (34.4) 34/12,759 (0.27) 90/36,314 (0.25) 37/12,210 (0.30) 57/26,107 (0.22) 29/ 11,470 (0.25)
66/6,483 ( 1.O) 244/7,246 (3.8) 72/9,748 (0.74) 86/2,510 (3.4) 153/12,759 (1.2) 224/36,314 (0.62) 109/12,210 (0.89) 130/26,107 (0.50) 65,' 11,470 (0.57)
Ochre white/ all
Xb&/ XbC/ light blue
18/28' (64) 52/66' (79) 5 / 16d (31) 6/6" (100) 0/24f None 2/2gg (7) 3/33)' (9) 3/39i (8) 1/ 20' (5)
0/20 None 0/8k None 0/15 None n.d.
XbuIochre/total" (%)
(0.34) (3.4) (0.02) (34)
0/50 None 0/10 None 0/25 None 0/20 None 0/20 None
None None None None None
Values in parentheses are percentages. 'Numbers in this column are products of the numbers in the columns labeled White plaques/total, Ochre whites/all white and XbuI-/white tested. Thus, the number in this columnis the fraction of the total yield for which there is evidence of accurate information transfer from RNA residues. 'The 18 phages in the numerator that were XbuI- were also ochre-suppressible. Five phages in the denominator that were ochre-suppressible were XbuI+, as were five that were not ochresuppressible. Two of the XbuI- phages in the numerator were not ochre-suppressible; 14 phages in the denominator were XbuI' and none of these were ochre-suppressible. Two of the five XbuI- phages in the numerator were ochre-suppressible. One of the five ochre-suppressible phage testedwas XbuI+. "All six phages tested for XbuI were ochre-suppressible. 'The five ochre-suppressible phages were included in the 25 tested for XbuI. RThetwo phages in the numerator were null for both XbuI and Hind111 and were deleted for both sites. Of the 29 phages in the denominator, two others were deleted but still XbuI'. Three phages that were suppressible were XbuI'. These three were not suppressible and harbored deletions covering the polylinker; they cut with neither Hind11 nor XbuI. 'The three XbuI- phages in the numerator were not suppressible; they were deleted for XbuI and also HindII. None of the ochre-suppressible phages were X b d - . I The one XbuI- phage in the numerator was also HindIII- and detected. Ten ochresuppressible phages were tested and all were XbuI+. One of the eight phage in the denominator had several XbuI sites but did not have an apparent insertion. primer RNA into the daughter strand and its use as a template in the next round of replication should also be considered. Short-patchreversetranscription (SPRT)seems an appropriate name for the phenomenon described in this study. The phrase has its etymology in short-patch mismatch correction in which only a few nucleotides are excisedconcomitant with mismatchrecognition and correction (LIEB1985; SICARDet al. 1985). Reverse transcriptionreferstothetermfor RNA-templated 1970; TEMINand MIZUTANI DNA synthesis (BALTIMORE 1970). The implication of SPRT is that in vivo DNA polymerases use a running start ona DNA template to traversea few ribonucleotidesunderconditionsin which the polymerase does not manifest bona $de reverse transcriptase activity. The reverse transcriptase responsible for branched RNA and DNA structures in
many bacteria, includingvarious strains of E. coli, is not present in E. coli K12 (LIMand MAAS1989), in which the present study was conducted. It has been suggested that single-stranded gaps in T4 DNA can be repaired via an RNA intermediate that is not subsequently excised but which acts as template for the next round of DNA synthesis (CUPIDO1983). The finding that p53 protein acts in vitro to promote both DNA and RNA annealing and strand exchange(OBEROSLER et al. 1993; BAKALKIN et al. 1994) prompted the suggestion that in vivo the excess RNA present in the nucleus could be used as a linker for joining DNA ends (BAKALKIN et al. 1994). Transformation of pneumococcus with a fraction vulnerable to both DNAse and RNase suggests that the transforming principle in these experiments could be an RNA-DNA chimera orhybrid (EVANS 1964). RNA transformation in Neurospora (MISHRAet
D. S. Thaler, G. Tomhline and
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FI(;I.RI. 2.-/1 representative gel. This gel is rcprcscntativc o f ' the analysis pcrformccl on man); tntttmts (scc Tahlcs I and 2 for data compilation) to determine whether a change had occurred a t the XhnI site. PCR amplitication of the h r 7 n region yielded a 2.2-kh segment, a sample of which was tested for restriction hv XbnI and separately by HindIII. Each PCR product is tested in three adjacent lanes. In the first of each set of three lanes, the PCR product was incuhated with the restriction huffer alone; in the second lane, with XhI; and in the third, with HindIII. The buffer and incuhation conditions in the three lanes were otherwise identical. Lanes 1, 5 and 50 contain lamhda Bs/EII markers (New England Riolabs). Lanes 2, 6, 9, 12, 1.5, 18, 21, 24 and 27 are PCR products incubated with huffer alone and contain the 2.2-kb fragment before restriction. Lanes 3, 7, 10, 15, 16, 19, 22, 25 and 28 contain thePCR product incubated with XbnI. In lanes 3, 16, 19, 22 and 25 the products were cuthy Xl,nI. Lanes 7, 10, 13 and 28 contain PCR products that were not cut hv XhnI. Lanes 4, 8, 11, 14, 17, 20, 23, 26 and 29 correspond similarly hut were incubated with HindIII; all were cut with HindIII.
nl. 1975) could be due to reverse transcription (M'ANG and LAMROWITZ 1993). Numerous mechanisms have been proposed in rclation to the context of adaptive or Cairnsian mutation. Onecommonaspect of theseproposals is thatthe physiology of cells under conditions of properlv perceived stress leads to an altered DNA metabolism that engenders alterations in the generation or fixation of mutation (FOSTER 1993; THAIXR1994). Proposed mechanisms of altered metabolism includereverse transcription (CAIRNS P/ nl. 1988; STEEIX and CAIRNS 1989), mismatch correction (STAHI.1988; HASTINGS and ROSENBERG 1992), homologous recombination (for re~iew see ROSENRERG 1994), and phage induction and transposition (SHAPIRO1984; FOSTERand CAIRNS1994; MAENHALIT-MICHEI. and SHAPIRO 1994). The incorporation of ribonucleotides into DNA as a consequence of properly perceived stress and their processing by SPRT may take its place among mechanismsproposedfor mutations arising under selection. M'e thank J. I,EI)EKRERG for his insight, encouragement and s u p port, and F. D(IKI'SC:II, P. F0SrF.R. R. ISFORI', J. L,I:.I>KRIWRG, S. LIL, B. M E S S ~ ~ E P. R , MODEL, E R ; I I I C ~ the Prokavotic Interest Group at Rockefeller University for sharing comments on the work and manuscript. The critiques of two anonymous reviewers were especially useful. This work was supported by grants from Procter RC Gamble Pharmaceuticals t o D.T. and by The Markev Foundation and Sloan Foundation to the 1;tboraton of Molecular Genetics and Informatics. K.Z. is a fellow o f the R. and 13. Sackler Foundation.
Nok n d d d in pro$ Mosrc PI (11. (1995) have found that Taq DNA polymerase can copy RNA if the polymerase gets a running start on a DNA template.
LITERATURE CITED Mr., 1.. EXQL'IST, R. Hor-IN.N. MURRAYand K. MCRKAY, 1983 Experimentalmethodsfor usewith lambda, pp. 433-466 in / . O I I I / ~/I, edited bv R. HESI)RIX, J. RORERTS,F. STAI~I. and R. M'EISIWRG. Cold Spring Harbor Press, Cold Spring Harbor, XY. B:\ut.KIs, G . . T. Y:\KOVI.F.VA,G . SEMVAXWA, IC P. MA(;S~'SSON, 1.. SZEKEI.Y c( d., 1994 p53 hinds single-stranded DNA ends and catalyes DNA renaturation and strand transfer. Proc. Natl. Acatl. Sci. USA 91: 413-417. BAI.II\IORE, D., 1970 RNA-dependent DNA polymerase in virions o f RNA tumour viruses. Nature 2 2 6 1209-121 I . BIASK,A , , J. A . G,\I.I-A\\-r,R.R. BURGESSand L. A. LOER,1986 An RNA polymerase mutant with reduced accuracy of chain elongation. Biochcm. 2 5 5920-5928. BowK,J. (;., K. RERESEK and T. A. KUNKEI., 1992 Uneqtaal human immunodeficienq virus type 1 reverse transcriptase error rates with RNA and DNA templates. Proc. Natl. Acad.Sci. USA 89:
AKwR,
6919-(i923.
BKO\\'S. I\'., R. W,vrsos, J. VIsoCRAl), IC TAIT,H. RoYER c/ nl., 1976 The structures and fidelity of replication of motlse mitochondrial DNA-pSCIOI I h R I recombinant plasmids grown in E coli K12. Cell 7: .5,17-530. CAIRSS, J., J. O\'ERBACGII AND S. MII.I.ER,1988 The origin of mutants. Natnre 3 3 5 142-145. C v r ~ n o M., , IS83 Bypass of pyrimidine dimers inDNAof hacteriw phage T4 via induction of primer RNA. Mutat. Res. 109: 1 - 1 I . DOIIIT, C., R. M'AGSEK and M. RADMAS, 198.5 Repair of defined single base-pair mismatches in fichm'rhin coli. Proc. Natl. Acad. Sri. USA 82: .503-50.5. E c ~ r o t sH., , and M. F. GOOI)MAN,1991 Fidelity mechanisms in DNA replication. Annu. Rev. Riochem. 60: 477-51 1. ERIE, D. A,, 0. H ~ ~ ~ l s ~ ~ ~ I ? ~M. . 4 C. \rA YOUS
