Báo cáo Y học: The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus pot

The processivity and fidelity of DNA synthesis exhibited
by the reverse transcriptase of bovine leukemia virus
Orna Avidan, Michal Entin Meer, Iris Oz and Amnon Hizi
Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
We have recently expressed in bacteria the enzymatically
active reverse transcriptase ( RT) of bovine l eukemia virus
(BLV) [Perach, M. & Hizi, A. (1999) Virology 259, 176–189].
In the p resent study, we have studied in vitro two features of
the DNA polymerase activity of BLV RT, t he processivity of
DNA synthesis and the fidelity of DNA synthesis. These
properties were c ompared with t hose o f the well-studied RTs
of human immunodeficiency virus type 1 (HIV-1) and
murine leukaemia virus (MLV). Both the elongation of the
DNA template and the processivity of DNA synthesis
exhibited by BLV RT are impaired relative to the other two
RTs studied. Two parameters of fidelity were studied, the
capacity to incorporate incorrect nucleotides at the 3¢ end of
the nascent DNA strand and the ability to extend these 3¢
end m ispairs. BLV RT shows a fidelity of misinsertion higher
than that of HIV-1 RT and lower than that of MLV RT. The
pattern of mispair elongation by BLV RT suggests that the
in vitro error proneness of BLV RT is closer to that of HIV-1
RT. T hese fidelity properties are disc ussed in the context of
the v arious retroviral RTs studied so far.
Keywords: bovine leukaemia virus; fidelity; processivity;
reverse transcriptase.
Bovine leukaemia virus (BLV) is a naturally occurring
exogenous B-cell lymphotropic retrovirus, which is the
aetiological agent of cattle leukosis. This disease is charac-
terized by an initial persistent lymphocytosis, which is
followed by the occurrence of clonal lymphoid B-cell

tumours a fter a long latency period [1]. T his virus is related
to human T-cell leukemia viruses type I and type II (HTLV-I
and HTLV-II, respectively), forming a subfamily of trans-
activating retroviruses [2]. The genomes of these c omplex
retroviruses have close to their 3 ¢ ends the r egulatory genes
tax and rex and the presence of both Tax and Rex proteins,
encoded by these genes, is required for viral replication.
These viruses also show nucleotide sequence similarities,
although BLV and HTLVs do not infect the same cell types,
because they probably bind different cell receptors [2–4].
The process of reverse transcription is the major early
intracellular event critical to the life cycle of all retroviruses.
The synthesis of the proviral DNA is catalysed entirely by
the reverse transcriptase (RT). The plus-strand viral RNA is
copied by the RNA-dependent DNA polymerase activity of
RT, producing RNA/DNA hybrids. The intrinsic ribo-
nuclease H (RNase H) activity of RT specifically hydrolyses
the RNA in these heteroduplexes. Finally, the plus-sense
DNA strand is synthesized by copying of the minus-sense
DNA strand by the DNA-dependent DNA polymerase
(DDDP) activity of RT [2,5]. As RT is a preferred target for
the development of viral inhibitors as antiretroviral drugs,
the structural and catalytic p roperties of R Ts have been the
focus of many recent studies, including three-dimensional
crystal studies [6–9]. A major effort was devoted to the
research of the RTs of the human immunodeficiency viruses
type 1 and type 2 (HIV-1 and HIV-2, respectively), which
are responsible for acquired immunodeficiency syndrome
(AIDS); most of the anti-AIDS drugs approved so far f or
the treatment of AIDS are inhibitors of the viral RT. Due to

the rapid emergence of drug-resistant HIV RT variants, the
development o f novel potent a nd specific inhibitors of HIV
RTs is still a principal objective in the chemotherapy of
AIDS [2,10,11]. Targeted drug d esigns rely on a better
understanding of the structure and function of retroviral
RT. Therefore, the investigation of RT of other retroviruses
should expand our understanding of the catalytic properties
of these closely related proteins.
We have recently expressed the recombinant RT of B LV
in bacteria. The gene encoding the RT was designed to start
at its 5¢ end next to the last codon of the mature viral
protease; namely, the amino terminus of the RT matches the
last 26 codons of the pro gene and is encoded b y the pol
reading frame [12]. BLV RT was purified and studied
biochemically: it exhibits all activities typical of RTs, i.e.
both R NA- and DNA-dependent DNA polymerases and
RNase H activity. Unlike most RTs, the BLV RT is
enzymatically active as a monomer even after binding a
DNA substrate. The enzyme s hows a preference for Mg
2+
over Mn
2+
in both its DNA polymerase and RNase H
activities. BLV RT was shown to have a relatively low
Correspondence to Amnon Hizi, Department of Cell Biology and
Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
69978, Israel. Fax: +972 3 6407432, Tel.: + 972 3 640 9974,
E-mail:
Abbreviations: BLV, bovine leukemia virus; HTLV, human T-cell
leukaemia virus; HIV, human immunodeficiency virus; MLV, murine

leukaemia virus, MMTV, mouse mammary tumour virus; AMV,
avian myeloblastosis virus; EIAV, equine infectious anaemia virus;
AIDS, acquired immunodeficiency syndrome; F
ins
, frequency of
insertion; F
ext
, frequency of extension; DDDP, DNA-dependent DNA
polymerase; RNase H, ribonuclease H .
Note: M. E. M eer and O. Avidan contributed equally to th e research
described i n this manuscript. The results presented are in partial ful-
filment of a PhD thesis (M.E.M.) at Tel A viv University.
(Received 6 August 2001, revised 14 N ovember 2001, accepted 3
December 2001)
Eur. J. Biochem. 269, 859–867 (2002) Ó FEBS 2002
sensitivity t o nucleoside triphosphate analogues, known to
be potent in hibitors of other RTs such as that of HIV [12].
In the present study we have extended the investigation of
BLV RT by a n in vitro analysis of the processivity and the
fidelity of DNA synthesis (namely, the ability of BLV RT to
misincorporate nucleotides at the 3¢ end of the growing
DNA strand and the further extending of the p reformed
mismatch es).
MATERIALS AND METHODS
Recombinant RTs and DNA polymerase activities
Monomeric BLV RT was expressed in bacteria and purified
to homogeneity after modifying the method published
recently [12] These modifications were as follows: (a) the
pUC12N6H BLV RT-transformed Escherichia coli were
grown in Terrific Broth (without glycerol) instead of

NZYM medium; ( b) the carboxymethyl Sepharose column
buffer was at pH 6.5 (instead of pH 7.0); (c) after the
purification had been carried out the BLV RT was further
concentrated in an Amicon Centriprep 30 concentrator.
Recombinant heterodimeric HIV-1 RT was expressed in
bacteria as described [13]. Recombinant murine leukaemia
virus (MLV) RT was also expressed in E. coli [14]. The
recombinant proteins containing six histidines at their
amino termini were purified as described above for BLV
RT, except for the f act t hat a ll buffers used to purify MLV
RT included 0.2% (v/v) Triton X-100 (instead of 0.1%).
The DNA polymerase activities were assayed as described
previously [15]. One unit of activity was defined as the
amount of enzyme that catalyses the incorporation of
1 pmol d NTP into activated DNA (that served as t he
template-primer) in 30 min at 37 °C, under the assay
conditions. Similar BLV, HIV-1 or MLV RT DNA
polymerase activities were used in all experiments described,
using 0.1–0.5 lg RT protein (according to the specific
activities of the different enzymes).
Template primers
For the experiments of DNA primer extension and
processivity, we used single-stranded circular /X174am3
DNA (from New England Biolabs) as the DNA template,
which was primed with a 15-residue synthetic primer
(5¢-AAAGCGAGGGTATCC-3¢) that hybridizes at posi-
tions 588–602 of the /X174am3 DNA. The synthetic
template-primers used for the experiments of m isinsertion
and preformed mispair extension are shown in Figs 2 and 3.
For analysis of site-specific nucleotide misinsertion, a

synthetic 50-residue template (with a sequence derived from
nucleotides 565–614 of /X174am3 DNA) was primed with
the same 15-residue oligonucleotide used f or extension and
processivity. This primer hybridizes to the sequence at
positions 24–38 (in the 5¢fi3¢ direction) of the 50-residue
template DNA (Fig. 2). For the extension o f DNA from a
mispaired terminus, the set of template-primers used is
composed of the same 50-residue oligonucleotide template
used for the mis insertion studies (Fig. 3), primed with a set
of 16-residue oligo nucleotides (that h ybridize to the nucleo-
tides at positions 23–38 of the template). Four versions of
16-residue primers w ere used, each differing from the other
only at its 3¢-terminal nucleotide (Fig. 3). All primers used in
this study were labelled at their 5¢ ends with c[
32
P]ATP
(using T4 polynucleotide kinase) and were annealed to the
templates with a twofold molar excess of each template over
its primer as described previously [16].
DNA polymerization and processivity experiments
The reactions were conducted in a final volume of 12.5 lL
6.6 m
M
Tris/HCl, 4 m
M
dithiothreitol, 24 lgÆmL
)1
BSA,
6m
M

MgCl
2
(for BLV and HIV-1 RTs) or 1 m
M
MnCl
2
(for MLV RT), final pH 8.0, supplemented by the
/X174am3 template-primer at a final concentration of
30 lgÆmL
)1
. For processivity studies, the BLV, HIV-1 and
MLV RTs, at equal DNA polymerase activities, were
incubated with the annealed template primer for 5 min at
30 °C. In all polymerization experiments shown we used
0.3–2 pmol RT per reaction (depending on activity) and
Fig. 1. DNA primer-extension and processivity of DNA synthesis
exhibited by BLV, HIV-1 and MLV RTs. All r eactions we re p erformed
with the 15-nucleotide synthetic 5¢ end-labelled oligonucleotide prime r
and a twofold excess of the template single-stranded circular
/X174am3 p hage DN A. The sequence of the primer and the experi-
mental details are described in Materials and methods. The symbols
for the DNA synthesis experiments are as follows: (–) DNA extension
performed with no DNA trap; (+) DNA extension experiments
conducted in the presence of unlabeled DNA trap. Molecular mass
markers are HinfI-cleaved dephosp horylated double -strand ed
/X174am3 D NA frag ments (Promega) labelled with [c-
32
P]ATP at the
5¢ ends by polynucleotide k inase.
860 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002

 2.5 pmol of the template p rimer. The reaction mixtures
were divided into two, w ithout or with a DNA trap of a
large excess of unlabelled activated herring sperm DNA,
at a final concentration of 0.6 mgÆmL
)1
(prepared as
described previously [15]). All reactions were initiated
immediately afterwards by adding the four dNTPs, each at
a final c oncentration of 20 l
M
, followed b y incubation at
37 °C for 30 min. The reactions were s topped by a dding
an equal volume of formamide dye mix, denatured at
100 °C for 3 min, cooled on ice and analysed by
electrophoresis through 8 % polyacrylamide/urea sequenc-
ing gels in 90 m
M
Tris/borate, 2 m
M
EDTA pH 8.0, as
described previously [17]. The extension products were
quantified by densitometric scanning of gel autoradio-
grams and the amounts of primer extended were calcu-
lated.
Fidelity of DNA synthesis
For site-specific nucleotide misinsertion, we assayed dNTP
incorporation oppo site to the A residue at position 23 of
the template as described [16,18] (see also Fig. 2). The
32
P-5¢-end-labelled 15-residue primer was extended in the

presence of increasing concentrations of eithe r 0–1 l
M
of
the correct dNTP (dTTP) or 0–1 m
M
each of the incorrect
dNTPs (dATP, dCTP or dGTP). All dNTPs used were of
the highest purity available (Pharmacia) with no detectable
traces of contamination by other dNTPs. For mispair
extension (Fig. 3), elongation of
32
P-5¢-end-labelled
16-nucleotide primers was measured with increasing con-
centrations of dATP as th e only dNTP present (0–1 m
M
range for the mispaired AÆA, AÆCorAÆG termini or a
0–1 l
M
range for the AÆT correct terminus) [16,18].
Reactions for all kinetic analyses contained 14 m
M
Tris/
HClpH8.0,4m
M
dithiothreitol, 4 m
M
MgCl
2
and
24 lgÆmL

)1
BSA. The reactions were incubated at 37 °C
for either 2 min (for the correct incorporation or correctly
matched DNA elongation), o r 5 min (for misincorporation
or extension of formed mismatched DNA). Kinetic
reactions were p erformed with a n  10-fold molar excess
of template-primer over BLV RT to ensure steady-state
kinetics in the linear range. All reaction products were
analysed by electrophoresis through 14% polyacrylamide/
urea in Tris/borate and EDTA seq uencing gels, and band
intensities w ere quantified as described above. This allowed
the calculation of reaction velocities, i.e. the amount of
Table 1. Quantitative analysis of DNA primer-extension and relative processivity of DNA synthesis. The radioactivity in the DNA bands in all
polynucleotide length ranges were s ummed and then the valu es o btained were divided by the sums o f a ll extended an d u nextended primers (detected
in the phosphoimaging sc annin g of the g els as shown in Fig. 1). The values given are the extended p rimers in each product length range e xpressed as
percentages of the total amounts (all extended and unextended primers) of the DNA products. The calculations were conducted separately for gel
lanes o f reactions carried out in the absence or prese nce of an excess of the unlabele d DNA trap (see Materials and methods) . The overall e xtensions
in the presence of the DNA trap, divided by the comparable figures obtained with no trap present, yielded the relative processivity values expressed
as p ercentages (see t ext). The values are th e menas calculated from two independent experiments ( one of w hich is shown in Fig. 1) and the v ariations
were usually < 15%.
Product length
(nucleotides)
BLV RT HIV-1 RT MLV RT
Without trap With trap Without trap With trap Without trap With trap
16–50 12.3 30.2 14.9 8.9 10.2 46
51–100 15.4 1.4 13.3 7.5 8.1 25.8
101–200 27.3 1.0 20.3 6.2 18.9 0.3
200–700 5.6 0.1 27.6 12.8 35.4 0.3
Overall extension 60.6 32.7 76.1 35.4 72.6 72.4
Relative processivity 53.9 46.3 99.7

Table 2. Quantitative analysis of DNA synthesis and processivity after correcting for the relative length of the DNA products. The data shown were
derived from the same two independent experiments as in Table 1. Here, the data were evaluated after correcting f or the mean lengths of the D NA
primers extende d by the three RTs und er the assay conditions used. Th e c orrectio n fo r th e a ctual a mount of dNTP incorporation fo r a given DNA
product was achieved by multiplying the radioactivity in each 5¢ end-labelled polynucleotide product length class by the median of the number of
nucleotides added in each range ( i.e. 17 nucleotides for the 16–50 nucleotide r ange, 4 2 nucleotides for the 51–100 n ucleotide r ange, 92 nucleotides for
the 101–200 nucleotide range and 342 nucleotides for the 200–700 nucleotide range). After introducing these factors, all values are expressed (as in
Table 1 ) as p ercentages of the total amounts of all primers exten ded in each length class. The values shown are the means calculated from the sam e
two independent experiments as in T able 1.
Product length
(nucleotides)
BLV RT HIV-1 RT MLV RT
Without trap With trap Without trap With trap Without trap With trap
16–50 3.3 12.4 2.4 2.1 1.4 23.8
51–100 9.3 1.3 4.7 4.0 2.5 30.2
101–200 33.2 1.8 14.5 6.3 11.7 0.7
200–700 22.5 0.8 66.2 43.4 73.0 2.2
Overall extension 68.3 16.3 87.8 55.6 88.6 56.9
Relative processivity 23.9 63.5 64.2
Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 861
total
32
P-labelled primer extended per minute in the
conditions used. The V
max
and K
m
values were calculated
from the double-reciprocal linear plots of velocity vs.
dNTP concentrations [16,18].
RESULTS

We have analysed in vitro two basic properties of DNA
synthesis by BLV RT, i.e. processivity and fidelity, both in
comparison with the well-studied RTs of HIV-1 (represent-
ing a low fidelity RT from t he lentivirus subfamily of
retroviruses) and of MLV (representing a relatively high
fidelity RT from the mammalian type C retroviruses)
[16,19–21]. The assays were performed with template-
primers already used in our laboratory with other RTs,
allowing comparison of information. Similar to all RTs
studied so far, BLV RT was found to lack a 3¢fi5¢
exonuclease (proofreading) activity (data not shown),
thereby p ermitting direct kinetic analysis of primer-exten-
sion. Previous data show that BLV RT, like HIV-1 RT,
prefers M g
2+
over Mn
2+
[12]. Therefore, all assays carried
out with these RTs were performed in the presence of Mg
2+
ions. For MLV RT, we have evidence that overall extension
of primers by this R T i n t he presen ce of Mn
2+
is fa r better
than with Mg
2+
, whereas the fidelity of DNA synthesis by
MLV RT (both misinsertion and mispair extension; see
below) is similar with Mg
2+

or Mn
2+
(unpublished data).
DNA synthesis under processive and nonprocessive
conditions
The processivity of a DNA or RNA polymerase is d irectly
proportional to the length of the n ascent polymeric products
formed before the enzyme molecules dissociate f rom these
product molecules and rebind the same or other template-
primer molecules [17,22]. The extent of product elongation
in one cycle of synthesis (before the polymerase disassociates
from the growing strand) may depend on kinetic parameters
that affect binding, single nucleotide addition, translocation,
pausing, etc. It is apparent that retroviral RTs are far from
performing totally processive events (where the entire
template molecule is copied as a consequence of a single
binding event of the enzyme) [17]. Therefore, we have tested
the processivity of the BLV RT in comparison with the two
well-studied RTs of HIV-1 and MLV.
In the primer-extension assay, described in Fig. 1, we
used the heteropolymeric single-stranded /X174am3 DNA
Fig. 2. The pattern o f DNA mispair formation by BLV, HIV-1 and MLV RTs. The s ynthetic 50-nu cleotide t emplate w as ann ealed to the
32
P-5¢-end-
labelled primer. The primer was extended with equal DNA polymerase activities of either BL V RT, HIV-1 RT o r M LV RT in th e presence of 1 m
M
of a s ingle incorrect dNTP (i.e. C , G , o r A ) o r 1 l
M
of the co rrect dNT P (dT TP) as described in Materials an d metho ds. Th e leve l of m isinsertion i s
apparent from the elongation of the primer in the presence of the incorrect dNTP relative t o that in the presence of dTTP.

Table 3. Kinetic parameters for site-specific misincorporation by BLV
RT. The 15-residue c)
32
P-5¢-end-labe lled primer was hybridize d to a
fourfold mo lar excess of the 50-residue temp late derived from t he
sequence of nucleotides 565–614 of /X174am3 DNA (Fig. 2). In each
set of the kinetic experiments, the template-primer was incubated with
BLV RT in the presence of increasing concentrations of a single dNTP.
The o ligonucle otide products w ere analysed and described in Materials
and m ethods. The apparent K
m
and V
max
values were determined from
at least two independent experiments performed as described in
Materials and methods and in the text and the variations were usually
< 20%. The values o f relative f requency of ins ertion (F
ins
)werecal-
culated as described in th e text.
Pair or mispairs
formed K
m
(l
M
)
V
max
(%Æmin
)1

) F
ins
AÆT 0.004 25 1
AÆC 28 15.1 1/11 600
AÆG 45 4.5 1/62 500
AÆA 55 1.1 1/300 000
862 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
as the template, which is annealed to a synthetic 5¢ end-
labelled primer. The extension of the primer by the RTs
was carried out in the absence or presence of a DNA trap,
added to the reaction mixture after the RT is given the
opportunity to bind the template-primer and before
polymerization starts (see Materials and methods). As
the trap is added in a vast excess, only prebound RT
molecules a re allowed to extend the labelled primer. This
restricts the extension reaction to only one round of
synthesis, hence once RT falls off, it binds the trap and is
not capable of performing further rounds of extending the
labelled primer. As expected, all three RTs produce longer
DNA products when multiple rounds of synthesis are
allowed. All RTs used have been calibrated to have the
same DDDP activity using activated DNA as the substrate
(see Materials and methods). Yet, the extent of elongation
obtained with BLV RT with no trap present is substan-
tially lower than that w ith HIV-1 RT and MLV RT. Most
products generated by BLV RT are up to  150 nucleo-
nucleotides in length, whereas for the other two RTs the
majority of the products are substantially longer than
200 nucleotides. The primer-extension labelled products
were quantified and the extent of elongation was calculated

by two methods. In the first, we calculated the amount of
product as a percentage of the total radioactivity detected
(Table1).BothHIV-1RTandMLVRTshow,withno
trap present, overall extensions of 73–76% which is
significantly higher than that of BLV RT (61%). The
majority of the products of the former two RTs (28–35%)
are longer than 200 nt, whereas only 6% of the products
synthesized by BLV RT are lon ger than 200 nt. These
differences are more remarkable after quantifying the
products generated by introducing a correction of the
lengths o f t he polynucleotides synthesized (Table 2). In this
method the average lengths of products was taken into
account in the calculation, as by being 5¢ end-labelled all
oligonucleotides have the same level of label per molecule,
irrespective of their lengths. This method corrects f or the
actual amount of d NTPs incorporated per given product.
The figures calculated by this second method show also
that the overall extension of BLV RT is significantly lower
than the extension calculated for the other R T studied
(68% for the BLV RT and  88% for HIV-1 and MLV
RTs).
As expected, when a DNA trap is present and only o ne
round of DNA synthesis is permitted, all RTs synthesize
less, as well as shorter, product when compared with
multiple-round synthesis (Fig. 1). The analysis of the
processivity of DNA synthesis i n the presence of a DNA
trap suggests that BLV RT has a processivity that is
substantially different from that of HIV-1 and MLV RTs. It
is apparent that BLV RT produces very short products,
most o f them < 30 nucleotides in length. In c omparison,

HIV-1 R T synthesizes products that are not substantially
different in their length from those generated when multiple
rounds of synthesis were allowed. MLV RT s ynthesizes, in
the presence of the trap DNA, products that are shorter
than those produced without a trap (but longer than those
generated by BLV RT). The quantitative analysis of the
relative processivity depends on the method of calculation.
When the overall extensions were calculated by the first
method outlined above (Table 1) MLV RT shows a superb
processivity of almost 100%, whereas BLV RT has
substantially lower processivity (54%) which is somewhat
Fig. 3. The pattern of mispair extension displayed by the purified RTs of BLV, HIV-1 and MLV. The
32
P-5¢-end-labe lled 16-nucleo tide prim ers were
hybridized to the 50-nucleotide template, producing duplexes with 3¢-terminal preformed mismatches, where N at the 3¢ endofeachrepresentsthe
incorrect nucleotide (A, C or G) or the correct on (T). The primers were extended with equal DNA polymerase activities of BLV RT, M LV RT, or
HIV-1RT(asdescribedinthetextandinMaterialsandmethods)inthepresenceofeither1 m
M
dATP (when the mispaired tem plate-primers were
elongated) or 1 l
M
dATP (in the c ase where the AÆT paired substrate was extended).
Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 863
higher than that of HIV-1 RT (46%). However, by
calculating the level of extension after correcting for the
lengths of the products synthesized, the data obtained is
substantially different (Table 2). HIV-1 and MLV RTs
exhibit relative processivity values, which are practically
identical ( 64%) whereas BLV RT shows a much lower
processivity of  24%.

The fidelity of DNA synthesis
All our previous studies with a variety of retroviral RTs
have shown that the parameters for fidelity of DNA
synthesis in vitro (i.e. 3¢ end misinsertion and the extension
of the performed 3¢ end mispaired primers) depend primar-
ily on the sequences of nucleic acids copied, rather than
whether DNA or RNA templates were copied [16,18,20,23].
Subsequently, in the present study we have analysed DNA
templates as representing both DNA and RNA substrates.
The formation of 3¢ mispaired DNA. To study the fidelity
of misinsertion, we used an assay system that measures t he
standing-start reaction of 3¢ end misinsertion. This is
achieved by following the misincorporation of incorrect
dNTPs opposite the template A nucleotide, which corre-
sponds to position 23 i n the 50-nucleo tide template used, in
comparison with the incorporation of dTTP (see Fig. 2 and
Materials a nd methods). The elongation of the
32
P-5¢-end -
labelled 15-nucleotide primer was performed with either
1 l
M
of the correct dNTP (dTTP) or 1 m
M
of each of the
incorrect dNTPs. Fig. 2 shows the gel analysis of the
elongation products with the correct or incorrect dNTPs. It
is apparent that the general pattern of primer extension
obtained with BLV RT is quite similar to this with HIV-1
RT. There is an elongation of one nucleotide in the presence

of 1 l
M
dTTP with no significant further extension. The
highest extent o f misincorporation is observed with dCTP,
forming CÆA mispairs, which are elongated further creating,
in the case of BLV RT, CÆT mispairs followed by the correct
pairs CÆG (18 nt). In comparison, HIV-1 RT is capable of
elongating further the 18-nucleotide primers to 19 nucleo-
tides (with a CÆT mispair at the 3¢ end). With both BLV and
HIV-1 RTs, the extent of mispair formation with dGTP and
dATP (forming GÆAandAÆA m ispairs, respectively) is
lower than with dCTP. MLV RT shows, on the other hand,
a substantially lower level of misincorporation relative to
the other two RTs studied. The only s ignificant misincor-
poration by MLV RT is apparent with dCTP, forming CÆA
mispairs, with no significant further elongation of the
16-mer products with this mispair at its 3¢ end.
To quantify the capacity of BLV RT to form 3¢ end
mispairs, four separate sets of primer-extension reactions
were carried out and analysed. In each case, we used
increasing concentrations of a single dNTP, thereby
determining the standing-start rate of synthesis of the
correct pair vs. the three possible mispairs. We used a
range of dNTP concentrations always below 1 m
M
(to
obey steady-state kinetic conditions) and calculated the
radioactivity in g el bands relative to the total amounts of
primer present (both the unextended and the extended
ones). The rates of misincorporation (V ¼ percentage of

primers elongated per minute) were calculated as a
function of dNTP concentrations, as described in Mate-
rials and methods. The apparent K
m
and V
max
values for
each dNTP were all derived from the double-reciprocal
curves of the initial velocities of primer extension vs. the
substrate concentrations (data not shown) and are given
in Table 3. To calculate the frequencies of misinsertions
(F
ins
values) we used the method used previously
[16,18,19]:
F
ins
¼
ðV
max
=K
m
Þ
w
ðV
max
=K
m
Þ
R

where ( w) denotes the incorrect nucleotide (dATP, dCTP
or dGTP) and (R) is dTTP. As expected from the pattern of
primer extensions (Fig. 2), the highest F
ins
values calculated
for BLV RT is for dCTP (1/11 600, see Table 3), whereas,
the formation of AÆA mispairs is very rare (F
ins
 1/
300 000) a nd the value calculated for dGTP incorporation is
slightly higher (1/62 500). The parallel F
ins
values calculated
by us previously in the same assay system for HIV-1 RT
were: 1/3460–1/9000, 1/32 250–1/41 500 and 1/52 200–1/
75 000; and for MLV RT: 1/25 000, < 1/300 000 and
< 1 /300 000, all for the formation of AÆC, AÆG, and AÆA
mispairs, respectively [16,20].
Extension of preformed 3¢ end mispaired DNA. Misin-
sertion by itself is not su fficient to create stable site-specific
mutations, unless the terminally mispaired DNA is further
extended, leading to the fixation of the mistaken sequence.
Therefore, the efficiency of extending 3¢ preformed mis-
matched primers is an essential factor in determining the
fidelity of DNA synthesis exhibited by different polyme-
rases. We have evaluated the ability of BLV RT to extend
preformed 3¢ end mispaired 16-residue primers (AÆA, AÆC,
AÆG) by analysing the extension of these primers during
DNA polymerization in the presence of the next comple-
mentary dATP (as the only dNTP present). These standing-

start reactions were performed in c omparison to H IV-1 RT
and MLV RT analysed with the same mispair ext ension
reactions. T he gel analysis o f the extension p roducts shown
in Fig. 3 shows that BLV RT is capable of e longating all
Table 4 . The kinetics of the extension of 3¢ end matched o r pre formed
mismatched primer termin i by BLV RT. The
32
P-5¢-end-labelled
16-nucleotide primers were hybridized to a 50-nucleotide template
derived f rom the seque nce of nucleo tides 565–614 of /X174am3 D NA,
producing duplexes with a 3 ¢-terminal p aired (AÆT) or mismatched
(AÆC, A ÆGorAÆA) primers (Fig. 3). E ac h t emplate- primer was incu-
bated with B LV RT in the presence of increasing co ncentrations of
dATP. The products were analysed as described in the text. The
apparent K
m
and V
max
values were the means calculated from at least
two independent experiments and the variations were usually < 15%.
The relative frequency F
ext
valuesaretheratiooftherateconstants
(V
max
/K
m
) for the mispair divided by the ratio of the corresponding
constants for the paired AÆTterminus.
Pair or mispairs terminus K

m
(l
M
)
V
max
(%Æmin
)1
) F
ext
AÆT 0.037 29.9 1
AÆC 86 13.6 1/5,100
AÆG 42 10.2 1/3,400
AÆA 68 11.4 1/4,800
864 O. Avidan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mispairs to roughly the same extent. In comparison, HIV-1
RT shows a substantial preference in extending the AÆC
mispairs over the AÆAandAÆG mispairs. MLV RT shows
the same preference in extending the mispairs (AÆC>
AÆA>AÆG) although the extent of elongating these
mispairs is significantly lower than the extensions observed
with HIV-1 RT.
To study the kinetics we f ollowed primer elongation as a
function of increasing concentrations of dATP as the only
dNTP present (Table 4). The ratios of all extended products
were calculated relative to the total amount of the primers as
a function of dATP co ncentration. The relative extension
frequency (F
ext
) values are defined as apparent V

max
/K
m
values, calculated for the formed mismatches, divided by the
corresponding V
max
/K
m
values obtained for the correctly
paired terminus (AÆT). The results show that the apparent
K
m
values for t he extension of all three studied mispairs by
BLV RT are similar. As expected, the V
max
value calculated
for the corrected paired terminus is higher than those values
determined for the mispaired t ermini. Also expected is t he
finding that this RT exhibits K
m
values fo r the extension of
the AÆA, AÆCandAÆG m ismatches t hat a re much higher
than the comparable value calculated for the correct AÆT
pair. As the extension of all three mispairs is about the same
(Fig. 3) it is not surprising that the relative extension
frequencies calculated for all m ispairs are quite similar,
ranging from 1/3400 (for the AÆG terminus) to 1/5100 (for
the AÆC mispair). On the other hand the F
ext
values

calculated previously in the same assay system were for
HIV-1 RT, 1/17 500–52 000, 1/3900–9200 and 1/35 000–
45,000, for the formation of AÆA, AÆC, and AÆG mispairs,
respectively [16,20].
DISCUSSION
Polymerases are processive, i.e. they can attach to the
polymeric substrates and perform polymerization cycles
without intervening dissociations [21,24]. A total proces-
sivity of synthesis of either DNA or RNA is accomplished
when the entire DNA or RNA template is copied as a
consequence of only one polymerase-binding event. Previ-
ous studies with various RTs have shown that the enzyme
is not highly pr ocessive while synthesizing DNA
[17,18,25,26].
The primer-extension and processivity of DNA synthe-
sis experiments shown in Fig. 1 indicate that these features
of BLV RT are significantly d ifferent th an those of both
HIV-1 RT and MLV RT. The data were quantified by
two methods (Tables 1 and 2). It is apparent that BLV
RT has a processivity substantially lower than that of the
two other RTs studied. Even without an excess of
unlabelled trap DNA, BLV RT is not capable of
synthesizing significant amounts of product DNA longer
than  120 nucleotides, with strong pausings between 90
and 120 nucleotides. In comparison, HIV-1 and MLV
RTs synthesize a relatively large amount of longer product
DNA molecules of 200–700 nucleotides in length, and the
majority of the products are in this length range. This
difference between BLV RT and the two other RTs
suggests that BLV RT has weaker binding to the DNA

substrate than the other RTs studied. I t might also be that
the premature pausings observed with BLV RT are
sequence-dependent. It will be of interest to study other
sequences than th ose used h ere to identify any un ique
sequences that cannot be copied easily by BLV RT.
The processivity experiment was conducted with a large
excess of trap DNA to prevent r ebinding of RT molecules
to the nascent DNA. The extent of DNA synthesis w ith
BLV RT is low and most products are very short (Fig. 1
and Tables 1 and 2). This shows that these products,
generated under s ingle-cycle conditions were syn thesized by
those BLV RT molecules that were bound to the DNA
before the addition of the trapping agent (and were
dissociated from the growing chain after incorporating only
few nucleotides) suggesting a poor processivity of this RT.
The pattern of the processivity seen with H IV-1 RT is
entirely different. Despite exhibiting a moderate processiv-
ity, the distribution of the elongation products in the
presence of the trap D NA is very similar to that s een in its
absence (although, as expected, the total amount of product
generated with the trap is lower, only 46–64% of those
synthesized without the trap). This phenomenon might
suggest that those HIV-1 RT molecules that can withstand
dissociation are capable of c ompleting the synthesis (or
show a high ÔpersistenceÕ of elongation without further
dissociation). MLV RT shows a behaviour that is interme-
diate between the apparent features of BLV RT and HIV-1
RT. The products formed in the presence of t he trap DNA
are substantially shorter than those synthesized with no trap
present (though they are much longer than those synthe-

sized by BLV RT with the trap DNA). The variations
observed in t he experiment shown in F ig. 1 necessitated the
use of the two quantification methods, summarized in
Tables 1 and 2. BLV RT shows an overall processivity of
DNA synthesis, which is significantly lower than the values
calculated for both HIV-1 and MLV RTs (see Table 2).
Yet, based on the amount of primers extended in t he
processivity experiments, BLV RT is capable of extending
about the same amount of primers as HIV-1 RT ( 50%),
despite the very significant differences in the ÔpersistenceÕ of
elongation (see Fig. 1 and Table 1). MLV RT is capable
of extending many more primer molecules ( showing a value
of almost 100% of relative processivity). It is possible that
these results may vary slightly depending of t he sequence of
the DNA copied and the conditions used in the experiments.
None of the RTs studied so far have any 3¢fi5¢
proofreading exonuclease activities, thus, making RTs more
error p rone than other DNA polymerases with this activity
[5,16,18,27,28]. Yet, a comparison of the overall fidelity of
DNA synthesis exhibited by RTs from different retroviruses
reveals significant differences among them. It was reported
that the RTs of HIV-1 and HIV-2 are relatively more error
prone than other RTs, such as those of avian myeloblastosis
virus (AMV) or MLV [19,20,23,29,30], explaining the
extensive genetic heterogeneity of both HIV-1 and HIV-2,
which affects viral pathogenesis, the rapid emergence of
drug-resistant variants and, hence, the progression of AIDS
[2,10,11,31]. We have also found that the relatively low
fidelity of DNA synthesis exhibited by HIV RTs is shared
by the RT of equine infectious anaemia virus (EIAV), which

belongs to the lentivirus subfam ily of retroviruses [16]. In
general, the fidelity of DNA polymeras es results from the
combination of nucleotide insertion and extension (in
addition to the presence or absence of proofreading
activities). Base substitution mutations during reverse
transcription can arise from the incorporation of a
Ó FEBS 2002 Processivity and fidelity of BLV RT (Eur. J. Biochem. 269) 865
noncomplementary nucleotide at the 3¢ e nd of the nascen t
DNA strand, follo wed b y an extension of the preformed
mispair [32,33]. Therefore, using parameters of both the
capacity to misincorporate an incorrect nucleotide and the
ability to extend the preformed 3¢ mispairs, it was suggested
that the overall rates of the in v itro error proneness in the
RTs s tudied is as follows: l entiviral R Ts > AMV
RT > MLV RT [16,19,29,30,34]. A more recent study
carried out with the RT of m ouse mammary tumour virus
(MMTV) has shown some deviation between the efficiency
of misincorporating an incorrect nucleotide and the ability
to elongate such a mispaired DNA [18].
We have studied the error proneness of BLV RT using
the defined template-primers and steady-state kinetics
methods used previously in our laboratory to study various
RTs [16,18–20,23,29,34]. The misinsertion frequencies
observed with BLV RT show that the specificity of
mismatch formation is AÆT>AÆC>AÆG>AÆA, com-
patible with the pattern observed with the other RTs [16,18–
20,23,29]. This misinsertion is affected by a major increase in
the K
m
values and a less significant reduction in the V

max
values. The F
ins
values are somewhat different than those
observed previously with HIV-1, HIV-2 and EIAV RTs.
The fidelity of misincorporation of MLV RT is substantially
higher than both BLV and lentiviral RTs (Fig. 2) and
[19,23]. Therefore, the overall order of error proneness of
the retroviral RTs studied, based on the site-specific
misincorporation experiment, is lentiviral RTs > BLV
RT  MMTVRT>AMVRT>MLVRT.
As to the capacity o f BLV RT to extend preformed
mispairs, it is apparent from Fig. 3 and Table 4 that BLV
RT extends all mismatches studied (i.e. AÆA, AÆC, and AÆG)
to approximately the same extent. The enzyme can extend
the mispairs by only one correct nucleotide (A) with no
further extension by misincorporating A opposite to G. This
is in contrast with the pattern of elongation observed here
with HIV-1 and MLV RTs (Fig. 3) and previously by these
RTs and the RTs of HIV-2, EIAV, MMTV and AMV
[16,18,19,23,29]. With all other RTs the efficiency of
preformed mispair extension with the same mispairs was
found always to be in the order AÆC>AÆA P AÆG.
Moreover, all RTs except for BLV RT were capable of
extending the AÆC mispair beyond the addition of only one
A. This ind icates that, und er the assay conditions used, all
other RTs can incorporate A opposite to G at position 18.
This is true even for MLV RT which has the highest fidelity
of all RTs studied. The steady-state kinetics analysis of the
mispair extension by BLV RT shows that the V

max
and the
K
m
values are relative ly close for all mismatched substrates
(Table 4). Moreover, majo r discrimination c an be attributed
to the relatively large K
m
differences governing the extension
of matched vs. mismatched base pairs, with much smaller
differences in the V
max
values. The high frequency of
extending the studied mispairs by BLV RT, relative to our
previous results, puts this RT on t he top o f the list with
lentiviral RTs in the in vitro error p roneness of RTs in the
following order: BLV RT  lentiviral RTs > AMV
RT > MMTV RT > MLV RT. However, the fact that
the mispaired DNA can be extended by BLV RT by only
one nucleotide beyond the mismatched 3¢ end may explain,
at least in p art, why virions of BLV grown in culture show
a relatively low mutation rate per replication cycle [35].
If extension of mispairs stops after the addition of one
nucleotide also in vivo, there will not be synthesis of full-
length mutated DNA and the overall fidelity will be
relatively high. This may also explain the observed in vitro
reduced processivity of BLV RT. Obviously, other viral and
cellular factors may also contribute to the result reported for
virions.
In summary, BLV RT shows a significantly low proces-

sivity of DNA synthesis together with a low fidelity, making
BLV RT unique among retroviral RTs. It had been
suggested a lready for mutants of HIV-1 RT that there is
an inverse correlation between the fidelity a nd processivity
of DNA synthesis (i.e. that the enhanced fidelity of
misinsertion and mispair extension is associated with a
reduced processivity [36]). The results with BLV RT in t he
present study as well as with other mutants of HIV-1 RT
[17] do not support this theory.
ACKNOWLEDGEMENT
We thank H. Be rman for typing the manuscript.
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