Reverse transcriptase of moloney murine leukemia virus with RNA polymerase activity

This invention provides a polypeptide capable of polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or more deoxyribonucleotides and one or more ribonucleotides. This invention further provides a purified Moloney murine leukemia virus reverse transcriptase, wherein an amino acid corresponding to position 155 of a wild type Moloney murine leukemia virus reverse transcriptase is a valine. This invention also provides a method of polymerizing a nucleic acid molecule comprising one or more deoxyribonucleotides and one or more ribonucleotides comprising: contacting a polypeptide capable of polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or more deoxyribonucleotides and one or more ribonucleotides with deoxyribonucleotides and ribonucleotides under conditions permitting incorporation of deoxyribonucleotides and ribonucleotides into a nucleic acid molecule. This invention still further provides a method of converting a DNA polymerase into an RNA polymerase comprising: a) structurally modifying the DNA polymerase; b) contacting the structurally modified DNA polymerase of step (a) with deoxyribonucleotides and ribonucleotides under conditions permitting incorporation of deoxyribonucleotides and ribonucleotides into a nucleic acid molecule; and c) detecting polymerization of ribonucleotides in the nucleic acid molecule resulting from step (b), the detection of ribonucleotides in the polymerized nucleic acid molecule indicating that the structurally modified DNA polymerase has been converted into an RNA polymerase. This invention also provides a nucleic acid molecule polymerized by the above-described method of polymerizing a nucleic acid molecule comprising one or more deoxyribonucleotides and one or more ribonucleotides.

Throughout this application, various references are referred to within 
parentheses. Disclosures of these publications in their entireties are 
hereby incorporated by reference into this application to more fully 
describe the state of the art to which this invention pertains. Full 
bibliographic citation for these references may be found at the end of 
this application, preceding the claims. 
BACKGROUND OF THE INVENTION 
The traditional classification of nucleic acid polymerases as either DNA or 
RNA polymerases is based, in large part, on their fundamental preference 
for the incorporation of either deoxyribonucleotides or ribonucleotides 
during chain elongation. The refined structure determination of Moloney 
murine leukemia virus reverse transcriptase, a strict DNA polymerase, 
recently allowed the prediction that a single amino acid residue at the 
active site might be responsible for the discrimination against the 2'OH 
group of an incoming ribonucleotide. Mutation of this residue resulted in 
a variant enzyme now capable of acting as an RNA polymerase. In marked 
contrast to the wild-type enzyme, the K.sub.m of the mutant enzyme for 
ribonucleotides was comparable to that for deoxyribonucleotides. The 
results are consistent with proposals of a common evolutionary origin for 
both classes of enzymes, and support models of a common mechanism of 
nucleic acid synthesis underlying catalysis by all such polymerases. 
A key characteristic of nucleic acid polymerases is their traditional 
classification as either DNA or RNA polymerases, which is determined by a 
given enzyme's ability to selectively use either deoxyribonucleotides 
(dNTPs) or ribonucleotides (rNTPs) as substrates for incorporation into a 
growing chain (1, 2). This classification, however, may not be as 
fundamental as originally thought (3-5). Crystallographic studies have 
demonstrated that DNA and RNA polymerases have remarkable structural 
similarities (refs. 6-15, reviewed in ref. 16), even though they lack 
extensive primary sequence homology. Both have a characteristic protein 
fold forming a nucleic acid binding cleft, and a trio of carboxylic acid 
residues thought to participate directly in catalysis through two bound 
divalent metal ions. Steady-state analyses further support the notion of a 
common stepwise polymerization mechanism (17, 18). These observations 
suggest that it might be possible to convert a DNA polymerase into an RNA 
polymerase by relatively minor alterations in its structure. 
Reverse transcriptases (RTs), encoded by all retroviruses, play a defining 
role in the retroviral life cycle (refs 19 & 20; for reviews see ref. 21). 
The enzyme is responsible for the synthesis of a double-stranded linear 
DNA copy of the RNA genome, which is subsequently inserted into the host 
genome to form the integrated proviral DNA. The reverse transcription 
reaction is complex, requiring RNA-dependent DNA polymerase activity, 
DNA-dependent DNA polymerase activity, and an associated RNase activity 
specific for RNA in RNA:DNA hybrid form (22). Although the enzyme can copy 
either RNA or DNA templates, RT, like all DNA polymerases, can only use 
deoxyribonucleotides, and not ribonucleotides as substrates. Studies of 
the HIV-1 RT have permitted modeling of the position of the incoming 
nucleotide at the active site (23, 24), with .alpha.-helices C and E, and 
.beta.-sheet strands 6, and 9-11, setting the major topology of the dNTP 
binding site. A recently determined crystal structure of a catalytic 
fragment of Moloney murine leukemia virus (MMLV) RT at 1.8 .ANG. 
resolution has made it possible to visualize how such selectivity for 
deoxyribonucleotides might be achieved: the enzyme is proposed to 
discriminate against ribonucleotides through an unfavorable interaction 
between the aromatic ring of Phe-155 and the 2'OH of the incoming rNTP 
(ref. 14; see FIG. 1). Here we report that substitution of this residue by 
valine, as predicted, does indeed render the enzyme capable of 
incorporating ribonucleotide substrates into products. 
SUMMARY OF THE INVENTION 
This invention provides a polypeptide capable of polymerizing i) 
deoxyribonucleotides; ii) ribonucleotides; or iii) one or more 
deoxyribonucleotides and one or more ribonucleotides. 
This invention further provides a purified Moloney murine leukemia virus 
reverse transcriptase, wherein an amino acid corresponding to position 155 
of a wild type Moloney murine leukemia virus reverse transcriptase is a 
valine. 
This invention also provides a method of polymerizing a nucleic acid 
molecule comprising one or more deoxyribonucleotides and one or more 
ribonucleotides comprising: contacting a polypeptide capable of 
polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or 
more deoxyribonucleotides and one or more ribonucleotides with 
deoxyribonucleotides and ribonucleotides under conditions permitting 
incorporation of deoxyribonucleotides and ribonucleotides into a nucleic 
acid molecule. 
This invention still further provides a method of converting a DNA 
polymerase into an RNA polymerase comprising: a) structurally modifying 
the DNA polymerase; b) contacting the structurally modified DNA polymerase 
of step (a) with deoxyribonucleotides and ribonucleotides under conditions 
permitting incorporation of deoxyribonucleotides and ribonucleotides into 
a nucleic acid molecule; and c) detecting polymerization of 
ribonucleotides in the nucleic acid molecule resulting from step (b), the 
detection of ribonucleotides in the polymerized nucleic acid molecule 
indicating that the structurally modified DNA polymerase has been 
converted into an RNA polymerase. 
This invention also provides a nucleic acid molecule polymerized by the 
above-described method of polymerizing a nucleic acid molecule comprising 
one or more deoxyribonucleotides and one or more ribonucleotides.

DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a polypeptide capable of polymerizing i) 
deoxyribonucleotides; ii) ribonucleotides; or iii) one or more 
deoxyribonucleotides and one or more ribonucleotides. 
In a preferred embodiment of the invention, the polypeptide has an amino 
acid sequence encoding a portion of a reverse transcriptase wherein the 
amino acid corresponding to position 155 of the wild type reverse 
transcriptase is a valine. In another preferred embodiment of the 
invention, the polypeptide is a nucleic acid polymerase. In a further 
preferred embodiment of the invention, the nucleic acid polymerase is a 
reverse transcriptase. In a most preferred embodiment of the invention, 
the reverse transcriptase is a Moloney murine leukemia virus reverse 
transcriptase. 
As used herein a nucleic acid polymerase is defined as a polypeptide which 
incorporates either deoxyribonucleotides or ribonucleotides into a growing 
chain of a nucleic acid molecule under appropriate polymerization 
conditions. A nucleic acid polymerase may be a DNA polymerase or an RNA 
polymerase. Synthesis of a nucleic acid molecule using a nucleic acid 
polymerase occurs by extension of a primer. The primer can be either DNA 
or RNA. The primer is annealed to a template strand, which may be either 
DNA or RNA. The sequence of the nucleic acid polymerized [added] to the 
primer is determined by the template. For reverse transcriptases the 
template can be either RNA or DNA. 
The nucleic acid polymerase of the subject invention differs from known 
nucleic acid polymerases by the ability to polymerize a nucleic acid 
molecule product which comprises RNA, DNA, or a hybrid of RNA and DNA. 
The composition of the polymerized nucleic acid molecule product is 
determined by the substrates present for polymerization, i.e., 
ribonucleotides, deoxynucleotides, or a combination of ribonucleotides and 
deoxynucleotides, and the template used. The substrates are present in 
concentrations permitting polymerization by the nucleic acid polymerase. 
This invention provides a purified Moloney murine leukemia virus reverse 
transcriptase, wherein an amino acid corresponding to position 155 of a 
wild type Moloney murine leukemia virus reverse transcriptase is a valine. 
In a preferred embodiment of the invention the purified Moloney murine 
leukemia virus reverse transcriptase is capable of polymerizing i) 
deoxyribonucleotides; ii) ribonucleotides; or iii) one or more 
deoxyribonucleotides and one or more ribonucleotides. 
This invention provides a method of polymerizing a nucleic acid molecule 
comprising one or more deoxyribonucleotides and one or more 
ribonucleotides comprising: contacting a polypeptide capable of 
polymerizing i) deoxyribonucleotides; ii) ribonucleotides; or iii) one or 
more deoxyribonucleotides and one or more ribonucleotides with 
deoxyribonucleotides and ribonucleotides under conditions permitting 
incorporation of deoxyribonucleotides and ribonucleotides into a nucleic 
acid molecule. Appropriate nucleic acid synthesis conditions which permit 
incorporation of deoxyribonucleotides and ribonucleotides into a nucleic 
acid molecule are known to one of skill in the art and include, but are 
not limited to, the presence of appropriate buffers and appropriate 
concentrations of substrates for the nucleic acid molecules to be 
produced. Suitable substrates may be deoxyribonucleotides (dNTPs), 
ribonucleotides (rNTPs), or a mixture of dNTPs and rNTPs. 
In a preferred embodiment of the above-described method of polymerizing a 
nucleic acid molecule comprising one or more deoxyribonucleotides and one 
or more ribonucleotides, the polypeptide has an amino acid sequence 
encoding a portion of a reverse transcriptase wherein the amino acid 
corresponding to position 155 of the wild type reverse transcriptase is a 
valine. In a further preferred embodiment the polypeptide is a nucleic 
acid polymerase. In a still further preferred embodiment the polypeptide 
is a reverse transcriptase. In a most preferred embodiment the reverse 
transcriptase is a Moloney murine leukemia virus reverse transcriptase. In 
another preferred embodiment of the above-described method the polymerized 
nucleic acid molecule is a ribozyme. 
The nucleic acid products may be any RNA molecule, any DNA molecule or a 
hybrid molecule of RNA and DNA. The nucleic acid sequence of the 
polymerization product is determined by the template nucleic acid strand 
which the nucleic acid polymerase uses to add bases, e.g. dNTPs, rNTPs or 
both dNTPs and rNTPs, to the primer which is annealed to the template. 
In a further preferred embodiment of the above-described method the 
polymerized nucleic acid molecule has at least one specific cleavage site. 
For example, a polymerized nucleic acid molecule may be comprised of DNAs 
with only one ribonucleotide polymerized within the nucleic acid strand. 
Such a hybrid nucleic acid molecule can be cleaved at the ribonucleotide 
under alkaline conditions. Accordingly, nucleic acid molecules with a 
specific cleavage site may be synthesized by the above-described nucleic 
acid polymerase. Alternatively, nucleic acid molecules having more than 
one specific cleavage site may also be produced using the above-described 
nucleic acid polymerase. The nucleic acid sequence is determined by the 
template used by the nucleic acid polymerase and the substrates used in 
the polymerization. 
This invention provides a method of converting a DNA polymerase into an RNA 
polymerase comprising: a) structurally modifying the DNA polymerase; b) 
contacting the structurally modified DNA polymerase of step (a) with 
deoxyribonucleotides and ribonucleotides under conditions permitting 
incorporation of deoxyribonucleotides and ribonucleotides into a nucleic 
acid molecule; and c) detecting polymerization of ribonucleotides in the 
nucleic acid molecule resulting from step (b), the detection of 
ribonucleotides in the polymerized nucleic acid molecule indicating that 
the structurally modified DNA polymerase has been converted into an RNA 
polymerase. 
In preferred embodiment of the above-described method the structural 
modification of the DNA polymerase in step (a) is performed by a point 
mutation or by polymerase chain reaction (PCR). In another preferred 
embodiment of the above-described method the point mutation or the 
polymerase chain reaction substitutes an amino acid corresponding to 
position 155 of a wild type DNA polymerase for a valine. 
Methods of altering the amino acid structure of enzymes, including DNA 
polymerases, are well known to one of ordinary skill in the art. These 
methods include, but are not limited to, point mutation and polymerase 
chain reaction, PCR, e.g. as described in Kunkel et al. Methods Enzymol. 
155, 166 (1987) and Ausubel et al. Current Protocols in Molecular Biology 
(1987), John Wiley/Greene. 
In a most preferred embodiment of the above-described method of converting 
a DNA polymerase into an RNA polymerase the DNA polymerase is a Moloney 
murine leukemia virus reverse transcriptase. 
This invention provides a nucleic acid molecule polymerized by the 
above-described method of polymerizing a nucleic acid molecule comprising 
one or more deoxyribonucleotides and one or more ribonucleotides. In a 
preferred embodiment the polymerized nucleic acid molecule has at least 
one specific cleavage site. In a further preferred embodiment the 
polymerized nucleic acid molecule is capable of digesting a nucleic acid 
molecule. In a most preferred embodiment the polymerized nucleic acid 
molecule is a ribozyme. 
This invention will be better understood from the Experimental Details 
which follow. However, one skilled in the art will readily appreciate that 
the specific methods and results discussed are merely illustrative of the 
invention as described more fully in the claims which follow thereafter. 
EXPERIMENTAL DETAILS 
Construction of RT Mutants. The RNase H-defective MMLV reverse 
transcriptase construct (RT-WT-H) has been described previously (25). 
RT-F155V-H was constructed by replacing a KpnI-SalI fragment of RT-WT-H 
(nucleotides 261-1108) with KpnI-AflII and AflII-SalI PCR-derived MMLV RT 
fragments. Primer F155V-sense 
(5'-ATATAGCTTAAGGATGCCGTTTTCTGCCTGAGACTCCAC-3') (SEQ ID NO. 1), bearing 
the mutant valine codon (in boldface type; nucleotides 463-465) and silent 
mutations creating an AflII site (underlined), and a downstream primer 
were used to generate the 0.2-kb AflII-SalI PCR fragment, while the 
F155V-antisense primer (5'-ATATAGCTTAAGATCAAGCACAGTGTACCA-3') (SEQ ID NO. 
2), bearing silent mutations to create an AflII site (underlined), and an 
upstream primer were used to generate the 0.6-kb KpnI-AflII PCR fragment. 
RT-F155Y-H, in which Phe-155 was substituted by tyrosine, was constructed 
by replacing the 0.2-kb AflII-SalI fragment of RT-F155V-H with a 0.2-kb 
AflII-SalI PCR fragment containing a TAT tyrosine codon and an AflII site 
introduced by the sense primer. 
Enzyme Purification. Recombinant RT enzymes were expressed in Eschericha 
coli DH5.alpha. and partially purified with DE52 resin as described (26) 
for use in homopolymer assays. For all other assays, enzymes were purified 
to near-homogeneity by chromatography on DE52 cellulose (Whatman), P11 
phosphocellulose (Whatman), and MonoS (Pharmacia) fast protein liquid 
chromatography (FPLC). 
Homopolymer Substrate Assays. Typical assays were performed using 
.apprxeq.40 ng of enzyme (as determined by immunoblot comparison with pure 
RT standards) in 50 .mu.l of RT reaction buffer (27) containing 60 mM 
Tris.HCl (pH 8.0), 75 mM NaCl, 0.7 mM MnCl.sub.2, 5 mM DTT, 12 .mu.g/ml 
homopolymer template, 6 .mu.g/ml oligonucleotide primer, 10 .mu.Ci/ml (1 
Ci=37 GBq) .sup.32 P-labeled nucleotide and 12 .mu.M unlabeled nucleotide 
substrate. 
Measurement of Enzyme Kinetics. Purified enzyme was added to substrates in 
reaction buffer to initiate the reaction. At each time point, 10 .mu.l of 
reaction solution was removed and stopped by addition of EDTA. Samples 
were spotted on DE81 paper (Whatman) and washed with 2.times. standard 
saline citrate, followed by scintillation counting. Radioactivity retained 
on the paper, in comparison with total radioactivity in each sample, was 
used to determine the amount of dTTP incorporated into the product. 
Parameters were determined by double reciprocal plot. 
Single Nucleotide Extension Assay. Oligonucleotide C14 
(5'-GGTTCCTACCGGCC-3') (SEQ ID NO. 3) was end labeled with 
[.gamma.-.sup.32 P]ATP using polynucleotide kinase (New England Biolabs) 
according to the manufacturer's specifications. The radiolabeled product 
oligonucleotide (*C14) was purified by G25 spin column (Boehringer 
Mannheim) and annealed to G17 (3'-CCAAGGATGGCCGGATC-5') (SEQ ID NO. 4) at 
room temperature for 0.5 hr. Primer extension was initiated by adding 3 
.mu.g of purified enzyme to 60 .mu.l of reaction buffer containing 60 mM 
Tris.HCl (pH 8.0), 75 mM NaCl, 0.7 mM MnCl.sub.2, 5 mM DTT, 0.1 .mu.M 
*C14/G17 and unlabeled nucleotide substrate at the indicated 
concentration. At each time point, 10 .mu.l of the reaction was taken out 
and mixed with 10 .mu.l stop solution (80% formamide, 0.1% xylene cyanol, 
0.1% bromophenol blue, and 0.1 M EDTA). The extension products were 
resolved by electrophoresis on a 23% urea polyacrylamide gel and detected 
by autoradiography. 
Ribonucleotide Incorporation by RT-F155V-H Using Heteropolymeric Templates. 
A 0.3-kb PCR fragment, generated by the F155V-antisense primer and the 
upstream primer, was cloned into pBluescript (Stratagene) by blunt-end 
ligation, oriented such that the antisense primer sequence was near the T7 
promotor in the vector. The plasmid was linearized and transcribed to 
generate a 0.32-kb RNA fragment by in vitro run-off transcription using T7 
RNA polymerase (Boehringer Mannheim) according to the manufacturer's 
instructions. A 0.3-kb single strand DNA was generated by asymmetric PCR 
using an excess of the F155V antisense primer. The upstream primer was 
annealed to either template, at concentrations of 40 nM primer and 50 nM 
template, and extended by 3 .mu.g of purified enzyme for 30 min at 
37.degree. C. in 60 .mu.l of reaction buffer containing 60 mM Tris.HCl (pH 
8.0), 75 mM NaCl, 7.5 mM MgCl.sub.2, 5 mM DTT, 500 .mu.M dNTPs, 1 unit/ml 
RNasin and 50 .mu.Ci of [.sup.32 P]rNTP. The extended products were 
precipitated, resuspended in 10 .mu.l stop solution and resolved by 
electrophoresis on a 5% urea polyacrylamide gel, followed by 
autoradiography. 
RNA Synthesis by RT-F155V-H. Primer oligonucleotide P17 
(5'AAGCCCCACATACAGAG-3') (SEQ ID NO. 5) was end labeled and annealed to 
template oligonucleotide T28 (3'-TTCGGGGTGTATGTCTCTGACAACCTGG-5') (SEQ ID 
NO. 6) as described above. The primer (0.1 .mu.M) was extended by 3 .mu.g 
of RT-F155V-H per 60 .mu.l using four dNTPs (500 .mu.M each) or rNTPs (500 
.mu.M each) as substrates. Products were processed and analyzed as 
described above. 
EXPERIMENTAL RESULTS AND DISCUSSION 
Examination of the active site of a high-resolution structure of the MMLV 
RT, and modeling of template-primer and substrate into the structure, 
resulted in a strong prediction that the 2'OH of an incoming rNTP would 
overlap with the bulky sidegroup of Phe-155 (14). To test the structural 
prediction, a mutant MMLV RT was constructed carrying a substitution of 
Phe-155 with valine in the hope that the smaller side chain would open the 
"door" of the enzyme to ribonucleotides (see Methods). As a control, a 
mutant was also generated in which Phe-155 was replaced by tyrosine, found 
at the corresponding position in HIV-RT and thought to play the same role 
as the phenylalanine in MMLV-RT. In anticipation of potential cleavages of 
the RNA products by the RNase H activity in MMLV-RT, the substitutions 
were introduced into an RNase H-defective MMLV-RT backbone (designated 
RT-WT-H), which contains a mutation from Asp-524 to Asn (28). RT-WT-H 
lacks almost all RNase H activity but retains DNA polymerase activity 
comparable to the wild type MMLV-RT (28). Each mutant enzyme was expressed 
in bacteria, partially purified, and assayed on homopolymer templates with 
various substrates. As predicted, the substitution of Phe-155 with valine 
rendered the enzyme (designated RT-F155V-H) capable of incorporating 
ribonucleotides into the products from two different templates (FIG. 2). 
The wild type enzyme was unable to use ribonucleotides, and the tyrosine 
substitution (designated RT-F155Y-H) did not alter the enzyme's behaviour. 
Of 20 substitution mutants with changes of unrelated residues also located 
near the active site, none showed similar effects (data not shown). 
The effect of valine substitution was further analyzed by kinetic 
measurements of the enzyme activity using purified RTs and 
poly(rA)/oligo(dT) as template/primer (FIG. 3). The results are summarized 
in Table 1. RT-F155V-H was similar to RT-WT-H with respect to rate 
constants (K.sub.m) for oligo(dT) and dTTP, as well as maximum velocity 
(Vmax) using dTTP as a substrate. These similarities between RT-WT-H and 
RT-F155V-H in these parameters, along with their comparable enzymatic 
activities on various template/primers under different conditions (data 
not shown), indicate that the overall structure of RT-F155V-H was hardly, 
if at all, changed by the substitution. However, the affinity of 
RT-F155V-H for rUTP was dramatically increased. Whereas the K.sub.m of the 
wild type enzyme for rUTP was almost 50-fold higher than for dTTP, mutant 
RT-F155V-H displayed comparable K.sub.m values for rUTP and dTTP (Table 
1). These results strongly imply that Phe-155 is a key amino acid that 
dictates the selective binding of deoxyribonucleotides to the enzyme. 
Interestingly, the V.sub.max of RT-F155V-H for rUTP was only marginally 
changed compared with the wild-type, remaining .apprxeq.100 fold less than 
for dTTP. 
TABLE 1 
__________________________________________________________________________ 
Summary of kinetic parameters 
(DT).sub.12 dTTP rUTP 
Enzyme 
V.sub.max .mu.mol .multidot. min.sup.-1 .multidot. mg.sup.-1 
K.sub.m, nM 
V.sub.max .mu.mol .multidot. min.sup.-1 .multidot. 
mg.sup.-1 K.sub.m, .mu.M 
V.sub.max .mu.mol .multidot. 
min.sup.-1 .multidot. mg.sup.-1 
K.sub.m, .mu.M 
__________________________________________________________________________ 
RT-WT-H 
1.02 .+-. 0.05 
5.27 .+-. 1.95 
0.38 .+-. 0.07 
9.2 .+-. 3.4 
1.17 .+-. 0.51 
443 .+-. 221 
RT-F155 0.81 .+-. 0.06 8.27 .+-. 3.44 0.41 .+-. 0.04 13.6 .+-. 2.4 5.45 
.+-. 0.53 4.34 .+-. 
__________________________________________________________________________ 
1.71 
The lower catalytic rate of RT-F155V-H for ribonucleotide substrates could 
be accounted for by either an inherently lower ribonucleotide 
incorporation rate, or a lower extension rate of a 
ribonucleotide-containing primer, or both. To distinguish between these 
possibilities, a single nucleotide extension assay was performed. As shown 
in FIG. 4a, the mutant extended .apprxeq.75% of the primer by addition of 
dTTP within 0.5 min, while even after 15 min, only .apprxeq.50% of the 
primer was extended by rUTP. In comparison, RT-WT-H incorporated barely 
detectable amount of rUTP even at a concentration as high as 200 .mu.M 
(FIG. 4b). Furthermore, the incorporation of rUTP slowed the catalysis of 
the following nucleotide, either DATP or rATP, .apprxeq.5 fold (FIG. 4c). 
Products with a 3' terminal ribonucleotide migrated slightly more slowly 
than those with a deoxyribonucleotide. 
To expand these kinetics analysis results, RT-WT-H and RT-F155V-H were used 
to copy single-stranded DNA or RNA templates using dNTPs or rNTPs as 
substrates (FIG. 5). RT-F155V-H incorporated all four ribonucleotides into 
long products from a mixture of rNTPs and dNTPs, using either DNA (FIG. 
5a) or RNA (FIG. 5b) as a template. In contrast, RT-WT-H could barely 
incorporate detectable ribonucleotides into the product, though it could 
synthesize DNA products efficiently. That RT-F155V-H had little preference 
for any particular ribonucleotide substrate supports the specific function 
of Phe-155 to distinguish ribonucleotides from deoxyribonucleotides. 
To further evaluate the ability of the enzyme to synthesize RNA, only 
ribonucleotides were used as substrates (FIG. 5c). Despite the slow 
ribonucleotide polymerization rate, which is consistent with the kinetics 
results, RT-F155V-H was able to make an extended product of pure RNA of at 
least 6 nt under the given conditions. It is worth noting again that the 
ribonucleotide products migrated slower than the corresponding 
deoxyribonucleotide ones, indicating that the ribonucleotide products did 
not come from possible dNTPs contamination in the rNTP preparations. 
The results presented above provide direct evidence supporting the common 
stepwise mechanism of nucleic acid polymerization underlying all 
nucleotide polymerases catalysis. Such a common catalysis mechanism, along 
with structural similarities among the polymerases (3, 11, 29), suggests 
that all nucleotide polymerases might have evolved from the same ancestor. 
Indeed, this notion is supported by the observation that point mutations 
in T7 RNA polymerase can result in the reciprocal change in specificity to 
that observed here; these mutations rendered the enzyme, normally specific 
for ribonucleotides, capable of incorporating deoxyribonucleotides (16, 
30). The mechanism by which these mutations act is unknown. The results 
also provide insight into how a DNA polymerase selectively uses 
deoxyribonucleotides as opposed to ribonucleotides as substrates. Multiple 
devices are employed by the enzyme to prevent incorporation of 
ribonucleotides, which might be lethal to organisms. In MMLV RT, Phe-155 
serves as a door to preclude ribonucleotides from binding to the enzyme; 
and ribonucleotides, once bound to the enzyme, are further discriminated 
against by catalytic machinery for both incorporation and extension. It 
would be interesting to identify sequences governing the catalytic rate of 
the enzyme to use ribonucleotide substrates, by structure-based 
mutagenesis or by colony screening following random mutagenesis (31). 
RT-F155V-H may prove to be a powerful tool to serve this purpose. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 6 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - ATATAGCTTA AGGATGCCGT TTTCTGCCTG AGACTCCAC - # 
- # 39 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - ATATAGCTTA AGATCAAGCA CAGTGTACCA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - GGTTCCTACC GGCC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - CCAAGGATGG CCGGATC - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - AAGCCCCACA TACAGAG - # - # 
- # 17 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - TTCGGGGTGT ATGTCTCTGA CAACCTGG - # - # 
28 
__________________________________________________________________________