From the
Human immunodeficiency virus (HIV) nucleocapsid protein (NC)
influences HIV reverse transcriptase (RT) catalyzed strand transfer
synthesis from internal regions of natural sequence RNA. In the strand
transfer assay reaction in vitro, primer synthesis initiated
on a donor template can transfer and be completed on an acceptor
template. NC was added at concentrations up to twice that needed for
100% template coating. As the concentration of NC was increased, primer
extension was stimulated until NC coated approximately 50% of the
template. Stimulation was caused in part by an increase in the number
of primers that sustained synthesis. Subsequent increments of NC
decreased synthesis. The presence of NC also increased the efficiency
of the strand transfer reaction, allowing a greater proportion of
extended primers to transfer from donor to acceptor templates.
Processivity of the RT on the donor template was measured using both
challenged and enzyme dilution assays. NC did not alter the proportion
of synthesis products that reached the end of the template, indicating
little effect on processivity. This result suggests that the increase
in full-length product synthesis, observed in reactions where the RT
repeatedly bound the primer-template, resulted from promotion of RT
reassociation by NC. Consequently, since the RT could not reassociate
with the template in the processivity assay, NC could not stimulate the
amount of full-length synthesis. No strand transfer was observed in
dilution processivity assays, suggesting that the RT must dissociate
and rebind during the transfer reaction. Stimulation of synthesis, e.g. by increased dNTP concentration, normally inhibits strand
transfer. Stimulation of both synthesis and transfer by NC indicates
that properties of NC that improve the transfer event prevail over the
negative effects of rapid synthesis on transfer efficiency.
The most accepted model of retroviral replication includes two
distinct DNA strand transfer reactions from the end of the viral genome
(Coffin et al., 1979; Gilboa et al., 1979; Temin,
1981). Strand transfer can also occur from internal regions of the
genomic RNA or complementary DNA in vivo (Hu and Temin, 1990a,
1990b). This reaction very likely contributes to the high frequency of
recombination observed for retroviruses (Preston et al., 1988;
Roberts et al., 1988; Huber et al., 1989; Hu et
al., 1990a). Internal strand transfer provides the virus with a
mechanism in which synthesis can proceed even if the HIV-RT
HIV-NC is a 55-amino acid protein derived by
proteolysis of the gag and gag-pol polyprotein
precursor (Veronese et al., 1987; Tritch et al.,
1991; Henderson et al., 1992). NC is a highly basic protein
that contains two zinc fingers of the form
Cys-X
NC also binds to other nucleic
acids, although less avidly. The order of binding affinities is
single-stranded RNA > single-stranded DNA > double-stranded DNA
(Surovoy et al., 1993; You and McHenry, 1993). The nucleic
acid binding site size of mature NC is 7 nucleotides (You and McHenry,
1993). The role of the zinc fingers in nucleic acid binding has been
studied. Mutation experiments show that the basic amino acids around
the zinc fingers, but not the zinc fingers themselves, are essential to
carry on the nucleotide binding by the protein (Lapadat-Tapolsky et. al., 1993; De Rocquigny et al., 1992). On the
other hand, at least one zinc finger is needed for RNA packaging (South et al., 1991; Gorelick et al., 1993; De Rocquigny et al., 1992). The role of the zinc ion in the reactions
involving NC is not yet well understood (Karpel et al., 1987;
Delahunty et al., 1992; Rice et al., 1993).
At
high concentrations, NC can form large co-aggregates consisting of NC
and nucleic acids (Lapadat-Tapolsky et al., 1993; Tsuchihashi
and Brown, 1994). It is believed that NC coats the viral RNA template,
possibly to protect it from cellular RNases or to stabilize unwound
strands in a similar fashion as single-stranded DNA binding protein
does in Escherichia coli (Kornberg and Baker, 1991). NC
promotes strand exchange from a double-stranded DNA to a
single-stranded DNA favoring the most stable duplex (Tsuchihashi and
Brown, 1994). NC has a complex effect on properties of nucleic acids
that is concentration dependent. At low concentrations, NC promotes
annealing of complementary DNA strands, while at higher concentrations
it promotes melting of double strands (Lapadat-Tapolsky et al., 1993; Tsuchihashi and Brown, 1994). NC was shown to stimulate the
annealing of the HIV R region sequences 3000-fold (You and McHenry,
1994). These findings argue for a possible role of NC in strand
transfer events, which necessarily involve both of these processes.
In this report we demonstrate that NC slightly enhances the
efficiency of internal strand transfer reactions by HIV-RT. At low NC
concentrations synthesis is stimulated. NC-promoted melting of
secondary structures is likely to contribute to this stimulation. At
higher concentrations, NC inhibits synthesis. We also demonstrate that
NC does not significantly alter the processivity of primer elongation
by the RT.
Recombinant HIV-RT with native primary structure was provided
by the Genetics Institute (Cambridge, MA). The enzyme has a specific
activity of 40,000 units/mg. One unit is defined as the amount required
to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37
°C using poly(rA)-oligo(dT) as template primer. HIV-NC was
chemically synthesized by the Louisiana State University Medical Center
Core Laboratories. The sequence of the mature NC used for synthesis was
that of the first 55 amino acids of the NC precursor protein described
by Khan and Giedroc(1992). The peptide was kept under reducing
conditions, and aliquots were stored in 10% 2-mercaptoethanol. The
peptide concentration was determined by quantitative amino acid
analysis, performed by the Cornell University Peptide Facility.
Identity of the peptide was confirmed by amino acid composition
analysis. A molecular weight of 6444 was determined by electrospray
mass spectrometry.
Aliquots of both HIV-RT and NC were stored at
-70 °C, and a fresh aliquot was used for each experiment. T4
polynucleotide kinase, T7 RNA polymerase, placental RNase inhibitor,
RNase-free DNase I, rNTPs, restriction enzymes, and G25 and G50
Sephadex (RNA) columns were obtained from Boehringer Mannheim; dNTPs
were from Pharmacia Biotech. Inc. Radiolabeled compounds and Nensorb 20
cartridges were from DuPont NEN. The 20-nucleotide-long DNA primer and
the 68-nucleotide-long DNA acceptor template were synthesized by
Genosys, Inc. (Houston, TX). All other chemicals were from Sigma.
Increasing concentrations of NC were added to the
strand transfer assay reaction described under ``Experimental
Procedures.'' Fig. 2shows that as the concentration of NC
was increased, the number of primers that started synthesis increased
by 11-79% as determined by PhosphorImager analysis of the total
amount of radioactivity in elongated primers. The stimulation of primer
initiation reached a plateau at NC concentrations that would coat more
than 50% of the template. With subsequent increments of NC addition,
the number of primers that underwent synthesis declined.
We define
transfer efficiency as the quantity of DNA primers extended to the end
of the acceptor template (T), divided by the sum of the
quantity extended to the end of the donor template (F) plus
those extended to the end of the acceptor (T). The quantities
of these synthesis products on the gel were determined with the
PhosphorImager. shows the transfer efficiencies calculated
for each reaction shown in Fig. 2. The efficiency of transfer was
slightly increased in the presence of NC. This increase was concomitant
with changes in band intensity in the region of homology between donor
and acceptor. A slight increase in transfer efficiency occurred even at
concentrations of NC that inhibit primer initiation. At high
concentrations of NC, the absolute number of primers initiated was
reduced. However, the proportion of initiated primers that underwent
full-length synthesis over both donor and acceptor templates was higher
than when NC was not present. Therefore, in addition to altering the
number of primers that initiate, NC influences transfer efficiency.
In order to
examine the effect of NC on RT processivity we performed strand
transfer reactions in the presence of a compound that traps and
inactivates RT that is not bound to polynucleotide substrate. A
suitable trapping compound is heparin (Peliska and Benkovic, 1992). The
appropriate concentration of heparin trap was chosen so that if the
heparin is mixed with the primer-template and then the RT is added, no
subsequent synthesis is observed. We used the minimal concentration of
heparin that would serve as a complete inhibitor of unbound RT. During
the course of the experiment, the primer-template was incubated with
the RT first, and then the reaction was started by adding
Mg
We also
studied the effect of NC on RT processivity in the absence of a trap.
This was accomplished by performing RT dilution experiments (Fig. 6). We diluted the RT to a level of 1 RT/20 templates. At
this level of RT, 90% of the primers do not sustain any synthesis
during the course of the reaction (Fig. 6, lanes6, 12, and 18). Consequently, it was
unlikely that after completion of one round of synthesis on a
primer-template, an RT would have returned to the same primer-template
during the time of the reaction. This is because unreacted
primer-templates are in large excess over RTs. Therefore, the lengths
of extension of primers that have undergone synthesis represent
elongation from a single RT binding event. At this RT dilution, the
distribution of the products of synthesis was the same in the presence
and absence of NC. PhosphorImager analysis of all the extended primers
shows that the same proportion of primers reached the end of the
template whether NC was present or not. Evidently, NC has no detectable
effect on processivity.
We have investigated the effect of HIV-NC on the
HIV-RT-catalyzed internal strand transfer reactions from a
heteropolymeric RNA donor template to a complementary acceptor
template. NC slightly enhanced the internal strand transfer reactions
from the template used. Previous work has correlated enhanced strand
transfer with reaction conditions that slow the progression of
synthesis through the region of homology between the two templates.
However, properties of NC suggest that it should melt secondary
structure in the template. NC is known to promote both melting and
reannealing of double-stranded DNA. These characteristics of the NC are
consistent with an increased rate of synthesis that occurs as the
concentration of NC is raised. If primers were elongated more rapidly
over the region of homology, the efficiency of transfer should have
been reduced. The observation of increased transfer indicates that
other features of the NC-coated template improve transfer efficiency.
This improvement is sufficiently large to overcome the effects of
faster primer elongation in the homologous region and results in a net
increase in transfer efficiency.
NC has a complex effect on
properties of nucleic acids, which is concentration-dependent.
Tsuchihashi et al. (1993) and Herschlag et al.(1994)
reported that at high concentrations, NC inhibited multiple turnover
and single turnover of a hammerhead ribozyme by shutting down the
cleavage step. At lower concentrations NC enhanced the multiple
turnover of the ribozyme by increasing the annealing of the substrate
RNA and by catalyzing the dissociation of the product after cleavage by
the ribozyme. Similarly, Tsuchihashi and Brown(1994) showed complete
melting of short double-stranded DNA at high NC concentrations, while
at lower concentrations, NC stimulated renaturation. You and
McHenry(1994) reported a very large stimulation of annealing of
complementary RNA and DNA segments of the HIV R region, which seems to
be caused by melting of secondary structure in these segments. Dib-Hajj et al. (1993) have evidence that two types of saturated
NC-polynucleotide complexes can be formed that differ in apparent site
size: n = 8 versusn = 14. The
smaller site size binding mode was also observed by You and
McHenry(1993). The formation of the smaller and larger site size
complexes occurs at higher and lower concentrations of NC,
respectively. The functional effects of NC that we have observed are
explainable by a progressive weakening of the stability of double
helices with increasing concentration of NC. However, a relationship
between site size and NC effects cannot be ruled out.
We have shown
that the effect of NC on synthesis stimulation is also
concentration-dependent. At low concentrations, NC increases RT
synthesis, while at high concentrations it inhibits synthesis. Part of
the stimulation is likely to result from the ability of NC to melt
secondary structures (Lapadat-Tapolsky et al., 1993;
Tsuchihashi and Brown, 1994; You and McHenry, 1994). We have observed
nonuniform changes in the pattern of paused intermediates in the
presence of NC. It is believed that the binding of NC to nucleic acids
is not specific with respect to sequence. The only evidence so far of
preferential NC binding to a specific sequence has been the interaction
with the putative HIV packaging (PSI) domain (Dannull et al.,
1994). This suggests that NC could also interact with other specific
viral sequences that have not yet been identified. Some sequence
specificity of binding could explain the nonuniform effect of NC on RT
pausing over different regions of the template.
Primer extension is
inhibited at high levels of NC. One possibility is that as the
concentration of NC approaches a value that can coat the majority of
the single-stranded polymer, large amounts of free NC become available
to displace the primer from the template.
Another explanation for
the inhibitory effect of NC is that it may promote the dissociation of
RT prior to initiation of synthesis from the primer template. NC forms
coaggregates, large complexes involving interaction with itself and
nucleic acids (Tsuchihashi and Brown, 1994; Lapadat-Tapolsky et
al., 1993). At high NC concentrations, excess protein could
aggregate at the recessed 3`-end and thereby hinder the binding of the
RT. Recent evidence argues in favor of this possibility. If the
primer-template-RT-dNTP complex is formed before NC is added to the
reaction, synthesis occurs and the NC stimulates melting of secondary
structures.
We determined that neither the
stimulatory nor the inhibitory effects of NC on RT synthesis were due
to an effect on RT processivity. At both tested concentrations of NC,
the proportion of primers that were extended to the end of the template
was the same as in the absence of NC. We obtained similar results
whether we used a heparin trap or diluted the RT enough to observe the
results of a single RT binding event. It appears from NC-induced
changes in product distribution under conditions where it stimulates
synthesis that NC melts some of the template secondary structures and
not others. Indeed, the large stimulation of annealing of R region
sequences suggests that NC is particularly effective at altering
secondary structure of this sequence (You and McHenry, 1994). On such a
template, processive synthesis may increase in response to NC. This
would be expected since the likelihood of the RT to dissociate from the
template is high at pause sites (Klarmann et al., 1993). The
results of our processivity assays in the presence of inhibitory levels
of NC suggest that NC does not inhibit synthesis by increasing the
dissociation of the RT from the template.
Examination of the time
course of primer extension in processivity assays shows little effect
of NC on the rate of movement of the RT over the template. However,
there is a large stimulation of synthesis of full-length products
observed in the standard assays in which the RT is allowed to carry out
multiple cycles of synthesis, dissociation, rebinding, and further
synthesis. This suggests that a step in the reaction that occurs in the
standard assay but not the processivity assay is being stimulated by
NC. A likely possibility is the rebinding of the primer-template. The
presence of the protein may create the appropriate environment to
facilitate nearby RTs to rebind the primer-template faster. Another
possibility is that nonspecific binding of the RT to portions of the
template away from the primer terminus is discouraged, allowing more RT
to be available for productive binding.
We propose that the
stimulation of strand transfer by NC is the net result of opposing
activities of this protein. On one hand, observed stimulation of
synthesis by NC should inhibit strand transfer. On the other hand,
strand exchange capacities of the NC should stimulate transfer.
Furthermore, we have presented evidence that NC improves the rebinding
of the RT to the template after dissociation, a process that should
also promote strand transfer.
The
concentration of NC necessary to coat 100% of all templates in the
reaction was calculated as 495 nM. The counts of all primers
elongated per reaction were determined by PhosphorImager and are shown
in the second row. The relative amounts of products that underwent
full-length synthesis over either the acceptor or donor templates are
shown, labeled T and F respectively. Transfer
efficiency is shown in the last row (for details see
``Results'').
We thank Drs. Jasbir Seehra and John McCoy,
representing Genetics Institute, for the generous gift of HIV-RT.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)encounters a gap on the genomic viral RNA. We have
previously demonstrated that HIV-RT is capable of catalyzing internal
strand transfer in vitro, from a donor template to a
homologous region on an acceptor template (DeStefano et al.,
1992). Internal strand transfer was found to be RNase H-dependent. It
also occurred most efficiently near positions where the RT is known to
pause during synthesis on the donor template. We also found that
decreasing the concentration of dNTPs in the synthesis reaction, so
that primer extension becomes slower, increases the efficiency of
strand transfer.
-Cys-X
-His-X
-Cys,
flanked by regions that are rich in basic residues. It binds to the
viral RNA, stimulates viral RNA dimerization (Weiss et al.,
1992; De Rocquigny et al., 1992; Prats et al., 1991),
and is essential for viral packaging and, therefore, infectivity
(Dupraz et al., 1990; Aldovani et al., 1990). NC also
enhances the annealing of the tRNA
primer to the viral
RNA template (Barat et al., 1989; Weiss et al., 1992;
De Rocquigny et al., 1992), presumably by promoting the
unwinding of the tRNA (Khan and Giedroc, 1992). This is consistent with
the observation that the presence of NC can enhance reverse
transcription in vitro (Weiss et al., 1992; De
Rocquigny et al., 1992).
Materials
Methods
Strand Transfer Reactions
The amount of HIV-NC
necessary to coat 100% of the templates used was calculated based on
one molecule of NC binding to every seven nucleotides (You and McHenry,
1993). NC was preincubated for 5 min with 0.5 nM
primer-template and 50 nM acceptor template (Fig. 1) in
50 mM Tris-HCl (pH8), 80 mM KCl, 1 mM
dithiothreitol, 0.1 mM EDTA (buffer A), 2% 2-mercaptoethanol,
and 1 mM rNTPs, all given as final reaction concentrations.
Then HIV-RT (0.4 units, unless otherwise specified) was added and then
incubated for 5 additional min. The reaction was started by adding
MgCl and dNTPs, in buffer A, to a final concentration of 6
mM and 100 µM, respectively (except where
indicated). In some reactions, heparin trap was also added at this
point, to a final concentration of 0.4 mg/ml. The NC concentrations
ranged from 0 to twice the concentration calculated to obtain a 100%
template-coating level, or from 0 to 990 nM, respectively. The
reactions were performed in a final volume of 10 µl, at 37 °C
for 30 min, unless otherwise specified. The reactions were terminated
by addition of an equal volume of 2
gel loading buffer (90%
formamide, 10 mM EDTA (pH 8), 0.1% xylene cyanol, 0.1%
bromphenol blue).
Figure 1:
Schematic representation of substrates
used in the strand transfer reactions. The donor template is 142
nucleotides long. It was primed with a 5`-P-labeled DNA
oligonucleotide 20 nucleotides long. The products of full-length
synthesis over the donor template are 108 nucleotides in length. The
acceptor template has an area of homology to the donor template of 27
nucleotides in the region indicated. The products of full-length
synthesis after transfer to the acceptor template are 118 nucleotides
long.
RNA/DNA Hybridization
A specific
20-nucleotide-long DNA primer was labeled with P at the
5`-end, using T4 polynucleotide kinase. The hybrids were prepared by
mixing the RNA donor transcript with the 5`-labeled DNA primer at an
approximately 1:10 ratio of 3`-termini in 50 mM Tris-HCl, 0.1
mM EDTA, 80 mM KCl buffer. This mixture was heated at
65 °C for 15 min and then cooled slowly to room temperature. The
primer-template complex was then passed through a G50 Sephadex (RNA)
column to remove unbound primers. The concentration of primer-template
was determined by measuring the
P specific activity
present in the labeled primer before and after passing the templates
through the column, assuming 100% hybridization. The purified
primer-templates were shown by native gel electrophoresis to contain
less than 1% unannealed primers.
Quantitation of Nucleic Acids
The concentration of
unlabeled RNA transcript was estimated by titration with a known amount
of 5`-P-labeled DNA primer (0.025 pmol), using the
conditions of the strand transfer reactions. The primer-templates were
either extended with murine leukemia virus RT or just separated on a
native gel. The concentration of RNA was estimated from the level of
RNA required to extend 50% of a known concentration of primers assuming
100% hybridization. The RNA concentration was also determined by
estimating the amount of RNA required to hybridize 50% of a known
concentration of radiolabeled primer. The concentrations of the DNA
primers and of the acceptor templates were determined by
spectrophotometry.
Quantitation of Primer Extension Products
All
quantitation was obtained with a PhosphorImager (Molecular Dynamics).
Runoff Transcript
T7 RNA polymerase-catalyzed
runoff transcription was performed as specified by the Boehringer
Mannheim manufacturer's protocol. Plasmid pBSM13()
(DeStefano et al., 1992) was linearized using BstN1
and then used for the runoff transcriptions. The transcript was treated
with RNase-free DNase I and was then gel purified by electroelution.
Gel Electrophoresis
Denaturing 8% sequencing gels
(19:1 acrylamide/bisacrylamide) containing 7 M urea were
prepared and subjected to electrophoresis as described (Sambrook et
al., 1989)
Effect of NC on Strand Transfer Reactions
NC has
been shown to promote annealing and melting of nucleic acids. These are
important steps in the strand transfer reaction. Initial experiments
addressed whether NC had an effect on internal strand transfer
reactions. We measured strand transfer using a donor substrate
consisting of an RNA segment to which a DNA oligonucleotide was
annealed (Fig. 1). Full-length extension of the primer on this
template resulted in a product 108 nucleotides in length. Also present
was a DNA acceptor template 68 nucleotides long, with a 27-nucleotide
region that was exactly homologous to the donor template beginning 31
nucleotides beyond the primer. Strand transfer and completion of
synthesis on the acceptor template resulted in a product 118
nucleotides long.
Figure 2:
Effect
of NC on the level of strand transfer. Reverse transcription was
performed for 30 min as described under ``Experimental
Procedures.'' NC concentration was titrated from that needed to
coat 200% of the templates (lane1), 100% (lane2), 50% (lane3), 25% (lane4), 12.5% (lane5), 6.2% (lane6), 3.1% (lane7), and 1.5% (lane8), to no NC (lane9). The length in
nucleotides of full-length donor template-directed DNA synthesis
products (F) and products produced by strand transfer events (T) are indicated. The region where the donor and acceptor
templates were homologous is indicated.
Of the
primers that initiated synthesis, the proportion that reached the end
of either the donor template or acceptor template was also increased as
the NC concentration was augmented (). However, the
proportion of initiated primers extended to pause sites in the region
of homology between the donor and acceptor templates was the same or
less in the presence of NC. This is consistent with the reported
ability of NC to melt secondary structures (Khan and Giedroc, 1992;
Tsuchihashi and Brown, 1994; You and McHenry, 1994).
Effect of Zn
NC has two Znon NC Simulation of
Strand Transfer
finger domains.
Some NC functions are not Zn
dependent, such as
increasing tRNA unwinding and stimulation of initiation of synthesis
(Khan and Giedroc, 1992; De Rocquigny et. al., 1992). The role
of the Zn
fingers or of Zn
ions
with respect to internal strand transfer reactions is not known. We
tested whether the presence of Zn
has an effect on
the stimulation of strand transfer by NC. We compared the effect of
ZnCl
on strand transfer reactions at different
concentrations of NC (Fig. 3). Note that the synthetic NC used
here is not initially chelated with Zn
. We observed
that NC continues to stimulate strand transfer in the presence of
ZnCl
. Furthermore, the concentrations of NC needed to
achieve maximum stimulation of strand transfer are slightly lower in
the presence of ZnCl
. A likely possibility is that the
presence of Zn
protects the NC from inactivation as a
result of denaturation. The effect would be to increase the
concentration of active protein. This interpretation is consistent with
the NMR observations, which show that NC is more stable in the presence
of ZnCl
(Morellet et al., 1992; Surovoy et
al., 1993). The remaining experiments discussed in this paper have
been performed both in the presence and absence of ZnCl
.
For brevity we are showing only those performed in its presence. In a
series of five independent experiments in the presence of ZnCl
strand transfer efficiency increased from 28 to 33% in reactions
with NC at 10% of template-coating level compared with reactions with
no NC. Application of a matched pairs t test indicated a
significance of this difference with p = 0.03.
Figure 3:
Influence of ZnCl on NC
activity. Reverse transcription was performed with decreasing
concentrations of NC in the presence or absence of ZnCl
(50
µM) as indicated. The length in nucleotides of full-length
donor template-directed DNA synthesis products (F) and
products produced by strand transfer events (T) are indicated.
Amounts of NC used were 200% template-coating level (lanes1 and 7), 100% (lanes2 and 8), 50% (lanes3 and 9), 10% (lanes4 and 10), 5% (lanes5 and 11), and none (lanes6 and 12).
Fig. 4presents a time course experiment of the transfer
reaction in the presence and absence of NC. Previous work has shown
that the standard transfer reaction is slower than synthesis over the
donor template, resulting in an increase in transfer efficiency with
time (DeStefano, 1994). Here transfer efficiency reached a maximum by
30 min, in the presence or absence of NC.
Figure 4:
Time course of strand transfer reaction in
the presence and absence of NC. Reverse transcription was performed in
the presence of 50 nM NC and in the absence of NC as indicated
in the figure. The reaction was stopped at different points in
time as indicated. The positions of full-length donor directed DNA
synthesis products (F) and transfer products (T) are
indicated.
Effect of NC on HIV-RT Processivity
Processivity
is the length of primer extension that occurs each time the RT
initiates synthesis and before it dissociates. Here primer extension is
not a simple distribution but a series of products extended to pause
sites and over the full length of the template. Consequently
processivity cannot readily be quantitated numerically. Instead we can
compare the distribution of products made by a single round of
processive synthesis. Fig. 2shows that NC at low concentrations
has a stimulatory effect on overall end product synthesis. This
suggests that NC at particular concentrations may increase the ability
of HIV-RT to remain associated with the template until full-length
synthesis is accomplished, increasing processivity.
, dNTPs, and the heparin trap. In this fashion we
were able to determine the length of products synthesized by the RT
during a single binding event. In the presence of ZnCl
, NC
stimulated synthesis at a 10% template coating level and was slightly
inhibitory at a 50% coating level (Fig. 3). The reactions were
performed at these two levels of NC. The products of synthesis were
resolved by gel electrophoresis (Fig. 5A). At the
stimulatory level of NC the total number of primers initiated was only
slightly higher than in the absence of NC. At the inhibitory level of
NC the total number of primers initiated was lower than in the absence
of NC. The time course progression of primer elongation was slightly
greater at the stimulatory level of NC. Interestingly, the positions of
pausing are distributed differently with the addition of NC. At the
highest NC level there was greater pausing over the first half of the
template and proportionally less in the homologous region than in the
absence of NC. However, the proportion of extended primers that
underwent full-length synthesis was the same with either concentration
of NC as well as without NC.
Figure 5:
A,
time course of primer extension in a challenged processivity assay.
Internal strand transfer reactions were performed as described under
``Experimental Procedures'' in the presence of a heparin
trap. Inhibitory (250 nM) and stimulatory (50 nM)
concentrations of NC were tested and compared with reactions in the
absence of NC (0 nM). The reactions were stopped at the
indicated intervals. LaneC represents a control
reaction in which the heparin trap was added to the reaction prior to
the RT. No synthesis occurred, indicating that the trap was effectively
inhibiting free RT from binding to the template. The same control
reaction shows no synthesis in the presence of NC (data not shown). The
positions of full-length donor-directed DNA synthesis products (F) are indicated. Products produced by strand transfer events (T) are not detectable. B, NC titration in the
presence and absence of a heparin trap. Strand transfer reactions were
performed in the presence () and absence (
) of heparin trap.
The radioactivity in full-length products of synthesis was determined
by PhosphorImager analysis. The amount of full-length product
synthesized in the absence of NC was used as a control. The amounts of
radioactivity in full-length products of synthesis using different
concentrations of NC are represented as percent of control (y axis). The different concentrations of NC utilized are indicated
as a percentage of template coating levels (x axis).
An effect of NC on processivity could
be masked if the heparin trap were competing away the NC from the
templates. We ruled out this possibility by performing strand transfer
reactions over a range of NC concentrations in the presence or absence
of heparin. At the concentration of heparin used, the stimulatory
effect of NC starts appearing at the same NC concentrations in the
presence and absence of heparin (Fig. 5B). This
demonstrated that the heparin was not altering the amount of NC bound
to the template. We note that NC concentration for maximum stimulation
varies somewhat between experiments (Tsuchihashi and Brown, 1994),
possibly because of differences in oxidation of NC aliquots.
Figure 6:
Dilution processivity assay. Amounts of RT
used were 2 units (lanes1, 7, and 13), 1 unit (lanes2, 8, and 14), 0.2 units (lanes3, 9, and 15), 0.1 units (lanes4, 10, and 16), 0.024 units (lanes5, 11, and 17) and 0.012 units (lanes6, 12,
and 18).
Requirement for RT Dissociation for Strand Transfer
Reactions
When reactions were performed in the presence of a
trap (Fig. 5) or with very diluted RT (Fig. 6) strand
transfer was very inefficient. The transfer product is slightly longer
than the full-length product on the donor template. Therefore, it is
possible but unlikely that the RT cannot synthesize that distance in
one round of processive synthesis. Instead, it is probable that
dissociation of the RT invariably accompanies the transfer process.
Reassociation of the RT would then be necessary for completion of
synthesis of the transfer product on the acceptor template. Since
reassociation is prevented under the conditions for measuring
processivity, essentially no full-length transfer product is observed.
Effect of dNTP Concentration on Internal Strand Transfer
in the Presence of NC
Internal strand transfer efficiency was
increased when concentrations of dNTPs were lowered from 50 to 1
µM (DeStefano et al., 1992). This concentration
of dNTPs slows synthesis and would be expected to have a similar effect
as pausing in the transfer zone. This suggests that pausing promotes
transfer simply by increasing the time that the RT and primer terminus
are in the homologous region. We investigated whether lowering the dNTP
concentration in the reaction would influence the effect of NC on the
strand transfer event. Once again we observed that at lower
concentrations of dNTPs strand transfer efficiency was improved (Fig. 7). Furthermore, stimulation of the transfer reaction
continues to be observed at lower dNTP concentrations. The stimulatory
action of NC should allow the RT to move faster through the region of
acceptor homology. The low concentration of dNTPs should slow movement
of the RT over the whole template including the homologous region. It
appears that the stimulation of movement of the RT over the transfer
zone by NC was counterbalanced by the low dNTP concentration. When the
RT movement was slowed by low dNTPs the increase in transfer efficiency
produced by NC was even greater than at standard dNTP concentration.
Figure 7:
Effect of dNTP concentration on strand
transfer reactions. The concentration of dNTPs was titrated from 150
µM to 0.2 µM. Strand transfer efficiency was
assessed by quantitating the products of synthesis over the donor and
acceptor templates using a PhosphorImager. Strand transfer efficiency
was calculated as described under ``Results.'' Strand
transfer efficiency was plotted against dNTP concentration. Strand
transfer in the presence () and absence (
) of NC was
compared.
(
)When NC is added before the complex
is formed, it inhibits initiation of primer elongation. It is possible
that the RT encounters difficulty in moving through the first few
nucleotides if NC is aggregated over the primer region. We are
presently studying this possibility.
Table: Quantitation of products of synthesis
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.