(Received for publication, August 26, 1994; and in revised form, December 16, 1994)
From the
The properties of recombinant p66/p51 human immunodeficiency
virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal
truncations in its p66 polypeptide were evaluated. Deletion end points
partly or completely removed -helix E` of the RNase H domain
(p66
8/p51 and p66
16/p51, respectively), while mutant
p66
23/p51 lacked
E` and the
5`-
E` connecting loop.
Although dimerization and DNA polymerase properties of all mutants were
not significantly different from those of the parental enzyme,
p66
16/p51 and p66
23/p51 RT lacked ribonuclease H (RNase H)
activity. In contrast, RT mutant p66
8/p51 retained endonuclease
activity but lacked the directional processing feature of the parental
enzyme. Despite retaining full endoribonuclease function, p66
8/p51
RT barely supported transfer of nascent(-)-strand DNA between RNA
templates representing the 5` and 3` ends of retroviral genome,
shedding light on the requirement for the endonuclease and directional
processing functions of the RNase H domain during replication.
In contrast to the DNA polymerase activity of human
immunodeficiency virus reverse transcriptase (HIV RT), ()antiviral drugs targeted to its ribonuclease H (RNase H)
domain are scarce(1, 2) , despite documentation that
this activity is essential for viral
infectivity(3, 4) . Lack of (i) detailed structural
information, (ii) defined substrates, and (iii) pure recombinant enzyme
for in vitro analysis have undoubtedly hampered past studies.
However, these issues have been largely resolved, thereby offering
RNase H activity as another avenue of therapeutic intervention in our
efforts to curtail the spread of HIV infection and devastating
consequences of acquired immunodeficiency syndrome (AIDS). For example,
the three-dimensional structure of HIV-1 RNase H is now available as an
isolated domain (5) and a component of the parental p66/p51
heterodimer (6, 7) . Furthermore, the availability of
synthetic RNA now allows construction of model substrates mimicking
steps in the replication cycle invoking RNase H activity, e.g. DNA strand transfer (8, 9) , generating the
polypurine tract primer for (+)-strand synthesis (10) and
removing the(-)- and (+)-strand primers from nascent DNA (11, 12) . These advances have benefited from
efficient methods of expression and preparation of recombinant HIV-1
and HIV-2 RT free of contamination by bacterial
enzymes(13, 14, 15) .
The observation that
an initial endonucleolytic cut is accompanied by processing of the RNA
template (although the latter was originally defined as exonuclease
activity(16) , we believe that the term ``directional
processing'' more accurately reflects these events), and the
ability of human and murine enzymes to hydrolyze double-stranded RNA
(RNaseH activity(17, 18, 19) ,
indicates an unexpected versatility for the 120-residue C-terminal
domain. Understanding this expanded repertoire of nuclease functions
should prove beneficial to drug development programs. Several lines of
evidence implicate structural elements at the extreme C terminus of the
RNase H domain in the architecture and activity of the p66/p51
heterodimer. Although disordered in the isolated HIV-1 and HIV-2 RNase
H domains(20) , the p66
5-
E` connecting loop
(residues 537-543) has been shown to interact with
H and the
I-
J connecting loop of the p51 thumb subdomain(21) .
The
5`-
E` connecting loop contains the invariant
His
, which has been shown for bacterial and retroviral
RNases H to play a role in substrate binding and
catalysis(22, 23, 24, 25, 26, 27) .
The last element of p66 RT,
-helix E` (residues 544-555),
together with p51 helices
H and
I, may also provide a
``floor and wall'' of the nucleic acid binding cleft
immediately adjacent to the RNase H catalytic center. (
)Finally,
helix E` contains the conserved
Asp
, which has been implicated by Davies et al. (5, 20) in divalent metal ion coordination. The
importance of these elements prompted us to analyze heterodimer RT
containing minor C-terminal truncations in its p66 subunit. Our
rationale was also based on analysis of reconstituted heterodimer HIV-1
RT containing a C-terminally deleted p51 subunit(28) .
Elimination of 13 p51 residues compromised tRNA binding in the
reconstituted heterodimer, while other functions were unaffected.
Extending the deletion end point a further 6 residues influenced both
the processivity of DNA synthesis and tRNA binding, while p51 RT
lacking 25 residues failed to dimerize.
Here, we purified and
evaluated heterodimer HIV-1 RT whose p66 subunit was truncated by 8
(p668/p51), 16 (p66
16/p51), or 23 residues (p66
23/p51).
In p66
8/p51 RT,
-helix E` lacks three residues, while the
entire
-helix (including the invariant Asp
) is
absent in p66
16/p51. p66
23/p51 RT lacks
-helix E` and
the
5`-
E` connecting loop, the latter of which contains the
invariant His
. Purified enzymes showed similar levels of
RNA- and DNA-dependent DNA synthesis on defined heteropolymeric
template-primers. However, while p66
23/p51 and p66
16/p51 were
devoid of RNase H activity, mutant p66
8/p51 retains
endoribonuclease function. Loss of directional processing activity
coincides with a sharp reduction in the efficiency with which mutant
p66
8/p51 mediates transfer of nascent DNA between RNA templates,
indicating its necessity at this step in retroviral
replication(29) .
Figure 1: Sequence of template-primers used for analysis of DNA- (A) and RNA-directed DNA synthesis (C). Both templates make use of the same 36-nt DNA primer and are also homologous over 31 bases immediately adjacent to the template-primer duplex. The positions at which incorporation of the respective ddNTP arrests DNA synthesis is indicated. B and D, intramolecular base-paired structures assumed by the single-stranded DNA and RNA templates, respectively. Structure B was derived by sensitivity to DNase I and S1, while RNase A, RNase CV, and S1 were used to derive structure D.
RNA-dependent DNA polymerase activity was determined in a similar manner on an analogous 90-nt template-36-nt primer, with the exception that the template was extended at its 5` terminus by 19 nucleotides. ``Programmed'' primer extensions were therefore identical in both cases, while full-length products from RNA- and DNA-dependent DNA synthesis were 90 and 71 nt, respectively. Reaction products were fractionated by high voltage gel electrophoresis through 10% or 12% polyacrylamide gels containing 7 M urea in Tris/borate/EDTA buffer(36) . After drying, gels were subjected to autoradiography, using the DuPont ``Reflections'' system.
Strand transfer reactions were performed in
a buffer of 50 mM Tris-HCl, pH 8.0, 75 mM KCl, 1
mM dithiothreitol, 0.1% Triton X-100, 7 mM MgCl, 100 µM dNTPs, 200 nM 5`-end-labeled 20-nt DNA/40-nt RNA, 480 nM acceptor RNA
template, and 200 nM p66/p51 HIV-1 RT. Reactions were
initiated at 37 °C by addition of RT; at the times indicated,
samples were withdrawn and DNA synthesis was terminated by
supplementing with EDTA to a final concentration of 110 mM.
Reaction products were fractionated by high voltage electrophoresis
through denaturing 15% polyacrylamide gels, visualized with a Molecular
Dynamics PhosphorImager, and quantified with ImageQuant software
(provided by the supplier).
Figure 2: Qualitative analysis of DNA polymerase activities of RT mutants. A, DNA-dependent DNA polymerase activity. For each panel, lanes a-d represent primer extension by 1, 4, 10, and 19 nucleotides, respectively, while lanes e represent DNA synthesis in the absence of chain termination. P designates the migration position of the unextended primer. B, RNA-dependent DNA polymerase activity. The P+1 extension reaction was omitted from this analysis. Lanes a-c represent primer extension by 4, 10, and 19 nucleotides, respectively, while lanes d indicate DNA synthesis in the absence of chain termination. Note the accumulation of unextended primer and stalled P+3/P+4 products in reactions catalyzed by wild type RT.
Comparison of wild type RT (Fig. 2A, panel
iv) and the three deletion mutants (Fig. 2A, panels i-iii) indicates that removal of as many as 23
residues from the RNase H C terminus was tolerated without any
noticeable alteration in the efficiency of DNA-dependent DNA synthesis. Fig. 2B illustrates RNAdependent DNA synthesis on a
related template-primer and provides a similar result. Analysis of the
RNA template with nuclease S1 (specific for single-stranded nucleic
acid), RNase CV (specific for double-stranded RNA), and RNase A (which
cleaves after pyrimidine residues in single-stranded RNA) indicate that
extensive intramolecular base pairing is adopted. ()However,
this presents no major impediment to the translocating enzymes,
evidenced by the lack of stalled intermediates. Curiously, the
exception to this is wild type p66/p51 RT, which (a) initiates
cDNA synthesis less efficiently and (b) experiences minor
stalling at the P+3 and P+4 positions. These features are not
reconciled by increasing the enzyme:substrate ratio (data not shown),
suggesting that initiation of RNA-dependent DNA synthesis may be
enhanced by minor incursions into the C-terminal RNase H domain. More
importantly, the data of Fig. 2B contradict previous
observations of Hizi et al.(30) , who determined that
the RNA-dependent DNA polymerase activity of their p66 mutant CT-23 was
4% of that derived from the wild type enzyme, while that of
mutants CT-16 and CT-8 was 45% and 60%, respectively. Retention of both
RNA- and DNA-dependent DNA polymerase function in heterodimer RT
containing these p66 subunits may reflect a more acceptable
conformation in the singly-mutated heterodimer as opposed to the
doubly-mutated, truncated homodimers of Hizi et
al.(30) .
Fig. 3A, panel
i, illustrates RNase H activity of wild type RT in the absence of
polymerization and in response to heparin challenge. In the absence of
heparin, where dissociation and rebinding is permitted, cleavage
products of 62-64 nt are evident, together with minor amounts of
a 71-nt RNA (lane 1). Assuming RT locates itself over the 3`
terminus of the DNA primer(9) , the 71-nt product implies
cleavage at position -17, while the 62- and 64-nt products
indicate cleavage at position -8 and -10, respectively.
Prebinding RT to template-primer, followed by addition of
Mg and heparin (which ``traps'' free and
dissociated enzyme, allowing a single binding event to be monitored)
reverses this pattern, i.e. cleavage at -17
predominates, while those at -8 and -10 are minimized (panel i, lanes 3-5). Since the 62-nt/64-nt
products fail to accumulate over an extended period of heparin
challenge (lanes 3-5), the data of Fig. 3A, panel i, indicate that initial
cleavage at position -17 is followed by directional processing of
the RNA template (by a second enzyme) to around position -8, at
which point it most likely dissociates from the DNA primer.
Figure 3:
RT mutant p668/p51 retains
endoribonuclease activity. A, determination of RNase H
activity on the 90-nt RNA-36-nt DNA template-primer of Fig. 1. Panel i, effect of heparin on RNase H activity in the absence
of DNA synthesis. All assays contained radiolabeled substrate in a
buffer lacking Mg
, which was added later to initiate
hydrolysis. Lane 1, RNase H activity in the absence of
heparin. Lane 2, preincubation of RT with heparin. In lanes 3-5, RT was incubated with template-primer prior
to addition of heparin and Mg
, and aliquots of the
reaction were analyzed after 30 s (lane 3), 2 min (lane
4), and 10 min (lane 5). OH, alkaline hydrolysis
ladder of the RNA template for determination of product lengths. Panel ii, RNase H activity of wild type and mutant RT in the
absence of DNA synthesis. Lane R, partial RNase A hydrolysis
profile of the single-stranded RNA template. Lane 1,
p66
8/p51 RT; lane 2, p66
16/p51 RT; lane 3,
p66
23/p51 RT; lane 4, wild type p66/p51. Panel
iii, RNase H activity of wild type and mutant RT following primer
extension by 10 nucleotides. Lane notations are as in ii. B, schematic representation of the RNase H activities of wild
type RT and mutant p66
8/p51 in the absence of DNA synthesis (upper) and following primer extension by 10 nucleotides (lower, indicated by the shaded portion of the DNA
primer). For both replication complexes, the endonuclease and
directional processing activities of wild type RT are designated by the arrows, while boxed arrows indicate the sites at
which p66
8/p51 RT functions as solely an
endoribonuclease.
In Fig. 3A, panel ii, template-primer was
incubated with each RT in the absence of dNTPs and heparin. While
p6616/p51 and p66
23/p51 RT failed to hydrolyze the substrate,
mutant p66
8/p51 retained significant activity. However, when
compared to the data of panel i, the products of this reaction
(71 and 69 nt) suggested endonuclease activity but an absence of
directional processing. This notion was supported by coordinating RNase
H activity with RNA-dependent DNA synthesis. Under conditions where the
primer was extended by 10 nt (Fig. 2B), wild type
enzyme gives rise to RNase H cleavage products between 63 and 53 nt (panel iii). Relocation of RT to the primer terminus of a
+10 replication complex implies RNase H hydrolysis between
positions -19 and -9. Although this RNase H hydrolysis
profile is complicated by molecules which fail to extend the primer and
others which stall at the P+3 and P+4 positions (see Fig. 2B), locating the extremity of RNase H cleavage at
position -9 is in keeping with RNase H function in the absence of
DNA synthesis. In contrast, the RNase H products in a +10
replication complex catalyzed by mutant p66
8/p51 (63 and 62 nt)
correspond to cleavage at -18/-17, again suggesting
predominantly endonuclease activity. Furthermore, the absence of
cleavage products arising from stalled enzymes is in keeping with data
of Fig. 2B, implying that the RT mutant p66
8/p51
traverses the RNA template more efficiently than the wild type enzyme.
Fig. 4A indicates components of a model strand transfer
system. Initially, RT extends a 20-nt DNA primer to the 5` terminus of
the 40-nt donor RNA template. RNase H hydrolysis of the latter
facilitates transfer of nascent, 40-nt DNA to a 41-nt acceptor RNA
template dictating further synthesis of a 61-nt product. A quantitative
evaluation of DNA strand transfer via accumulation of the 61-nt product
is presented in Fig. 4B. Following an initial lag, wild
type RT supports efficient transfer and synthesis of the 61-nt DNA
product. In contrast, strand transfer in a reaction catalyzed by mutant
p668/p51 is reduced to 3-4% and only after a considerably
longer lag (
10 min). Inspection of the original phosphorimage
indicated that the reduction in p66
8/p51 RT-catalyzed strand
transfer was not a consequence of reduced cDNA synthesis, since the
40-nt intermediate accumulated to the same level with both enzymes
(data not shown). The data of Fig. 4B rather suggest
that endonuclease activity of p66
8/p51 RT, upon reaching the 5`
terminus of the donor RNA template, generates a 17-nt RNA which remains
stably hybridized to nascent DNA. The requirement for directional
processing to complete DNA transfer implicates a specific role for this
RNase H function in facilitating transfer of nascent DNA between (or
within) strands of the retroviral genome during replication.
Figure 4:
Endonuclease activity of p668/p51 RT
fails to support DNA strand transfer. A, features of the
strand transfer system. The components of the assay (i)
comprise a 40-nt donor RNA template derived from the repeat (r) end of the HIV-1 genome which is primed with a 20-nt DNA
primer and a 41-nt acceptor RNA, derived from the u3 genomic sequence,
with homology to the last 20 bases of the donor template. Following
primer extension to the 5` terminus of the donor template (ii), a combination of polymerase-dependent and independent
RNase H activities allow release and transfer of nascent 40-nt DNA to
the acceptor template. Subsequent primer extension on the acceptor
template yields a 61-nt strand transfer product. B,
quantitative analysis of the strand transfer products derived from
reactions catalyzed by wild type p66/p51 HIV-1 RT (closed
symbols) and the deletion mutant p66
8/p51 (open
symbols). Product bands were visualized by phosphorimaging and
quantified using ImageQuant software supplied by the
manufacturer.
Figure 5:
DNase I footprinting of replication
complexes containing heterodimer mutants. A, protection of
primer nucleotides of +1 replication complexes. For these
experiments, primer DNA was labeled at its 5` terminus with P. Lane 0, control DNase I digest of
template-primer in the absence of RT; lane 1, wild type
p66/p51 RT; lane 2, p66
8/p51 RT; lane 3 p66
16/p51 RT; lane 4, p66
23/p51 RT. Protection
of primer nucleotide by wild type to position -24 correlates with
structural predictions of Nanni et al.(21) . Positions
on the primer where alterations are evident with mutant RT are
indicated with arrows. B, protection of template
nucleotides in +4 replication complexes. Lane 0, DNase I
digest of template-primer duplex. Template DNA was labeled at its 5`
terminus with
P. Lane E, DNase I digest of
template DNA on which the primer was extended by 4 nucleotides. Lane 1, wild type p66/p51 RT; lane 2, p66
8/p51
RT; lane 3, p66
16/p51 RT; lane 4, p66
23/p51
RT. While the upstream boundary of the replicating enzymes is evident
at position -22, interactions with the single-stranded template
to position +6/+7 is derived from data of Boyer et
al.(41) . C, protection of template nucleotides
in +10 replication complexes. Lane 0, control digest of
template DNA containing an unextended primer. Template DNA was labeled
at its 5` terminus with
P. Lane 1, wild type
p66/p51 RT; lane 2, p66
8/p51 RT; lane 3,
p66
16/p51 RT; lane 4, p66
23/p51 RT. Alterations to
replication complexes containing mutant RT at position -19 are
indicated. In panels B and C, the open arrow indicates the position at which the chain-terminating ddNTP was
added to the primer (see Fig. 1).
In Fig. 5A, interaction with the
DNA primer of the template-primer duplex (Fig. 1A) was
evaluated following its extension by a single nucleotide (+1
replication complex). In keeping with earlier data, ()wild
type RT protects as far as position -24 from hydrolysis (lane
1). Partial elimination of
-helix E` in mutant p66
8/p51
is accompanied by a moderate increase in DNase I sensitivity at
position -23 (lane 2). Removal of the entire
helix
in mutants p66
16/p51 (lane 3) and p66
23/p51 (lane 4) has the consequence that reactivity at positions
-24 and -23 is equivalent to that of the naked
template-primer duplex. The data of Fig. 5A thus
illustrate that
-helix E` of the RNase H domain is sufficiently
close to the template-primer duplex to afford protection from DNase I
digestion. Fig. 5B presents profiles of the template
strand in +4 replication complexes. While wild type enzyme
protects nucleotides -22 to +6/+7 (lane 1),
subtle alterations in the template-primer duplex at positions
-18/-19 are evident as the size of C-terminal deletion
increases (lanes 2-4). Limited information is available
from these complexes on interactions with the single-stranded portion
of the template. This is better illustrated in +10 replication
complexes (Fig. 5C), where RT advances to protect 6
template nucleotides forming a short hairpin structure near the 5`
extremity of the DNA template (Fig. 1A). While the
hydrolysis profiles at the leading edge of the replication complexes
are identical, reactivity at position -19 again increases with
the extent of C-terminal deletion. The combined data of Fig. 5thus suggest that the manner in which the RNase H domain
interacts with the template-primer duplex is influenced by stepwise
removal of
-helix E`, while at the same time contacts between the
N-terminal p66 fingers subdomain and the single-stranded template are
unaffected.
Using defined heteropolymeric substrates, we evaluated the consequence of short truncations into the HIV-1 RNase H domain on dimerization, DNA polymerase, RNase H, and strand transfer properties of the parental heterodimer. Although mutagenesis studies on RNase H are available(42, 43, 44) , these often use in situ assays in polyacrylamide gels. In addition to being difficult to quantify, such approaches fail to address the biologically relevant enzyme (the p66/p51 heterodimer) and cannot discriminate between a defective heterodimer or monomers which fail to dimerize, since RT functions are dependent on dimerization(45, 46) . Furthermore, the use of defined heteropolymeric substrates should be promoted, since assessing RNase H function as loss of acid-precipitable counts from a randomly-generated hybrid cannot assess whether the endonuclease or directional processing function is selectively impaired. This is well exemplified in a recent study by Post et al.(47) with murine leukemia virus RT altered in its connection subdomain or fused to E. coli RNase H.
Bearing these caveats in mind, we demonstrate here
that removing residues Ser-Leu
from the
RNase H domain of p66 RT yields a stable heterodimer which retains DNA
polymerase function, but displays only a subset of its RNase H
activities. Loss of directional processing is reflected by low levels
of DNA strand transfer, indicating its importance at stages in
replication involving strand transfer. Based on data from Peliska and
Benkovic(9) , we interpret our findings in terms of a mutant
capable of copying DNA to the 5` terminus of the RNA template, after
which polymerase-dependent RNase H activity yields a single
endonucleolytic cut at or around position -17. Loss of
directional processing ``locks'' RT mutant p66
8/p51 with
its DNA polymerase active center over the 3` OH of the fully-extended
primer. Should dissociation from template-primer occur, enzyme which
rebinds would again be oriented in a manner dictated by the primer
terminus(9) , thereby preventing template cleavage beyond
position -17. p66
8/p51 RT is thus ``anchored'' to
the terminus of the template-primer duplex. In the absence of
polymerization-independent RNase H activity(9) , which we
believe is analogous to the directional processing events shown here,
the level of strand transfer observed most likely reflects slow melting
of the residual 17-18-nt donor RNA fragment from nascent DNA.
While the ability to uncouple the two RNase H activities was
surprising, support for the dependence of strand transfer on
directional processing is provided by studies with the RNase
H-deficient enzyme p66
/p51(3) . In this
case, substitution of Mn
for Mg
restores endoribonuclease activity, but DNA strand transfer is
likewise inhibited. (
)
The crystal structure of the HIV-1
RNase H domain may provide an explanation for our findings. Davies et al.(5) propose that four invariant residues,
namely Asp, Glu
, Asp
, and
Asp
participate in coordinating two metal ions. Of these,
the backbone carbonyl oxygen of Asp
forms a hydrogen bond
with the side chain hydroxyl of Ser
of
-helix E`.
Since the 8-residue deletion in p66
8/p51 eliminates
Ser
, reduced constraints on Asp
may have
the consequence that binding of one divalent cation is either impaired
or eliminated. This notion is not unreasonable, since the model of
Davies et al.(5) predicts that Asp
and
Asp
coordinate one divalent cation (site B), while those
of Asp
, Glu
, and Asp
coordinate a second (site A). Thus, if a two-metal ion mechanism
of catalysis is correct(5) , loss of coordination at site B may
influence directional processing. Although the catalytic mechanism for
RNase H remains to be established, potential alterations to metal ion
coordination cannot significantly influence placement of the RNA-DNA
hybrid in the RNase H active site, since cleavage 17-18 nt from
the DNA polymerase catalytic center is preserved with mutant
p66
8/p51. Lack of stalled intermediates during RNA-dependent DNA
synthesis by this mutant may indicate that a truncated
-helix E`,
coupled with altered metal binding, affords the RNA-DNA hybrid more
flexibility following initial endonucleolytic cleavage, thereby
rendering directional processing sterically unfavorable.
If
structural predictions of Nanni et al.(21) are
correct, stepwise removal of -helix E` of the RNase H domain could
alter contacts to the template-primer duplex. Although enzymatic
footprinting offers limited resolution, it was capable here of
highlighting such alterations (Fig. 5), the most notable being
at positions -23 on the primer and -19 on the template.
These are rendered DNase-sensitive as the extent of C-terminal deletion
increases, supporting the notion that
-helix E` contributes to the
wall of the nucleic acid binding cleft, downstream of the RNase H
active center.
Although we must interpret DNase I
footprinting data with caution, it is interesting to note that
interactions of the N-terminal fingers subdomain with template
sequences preceding the DNA polymerase active center are unaffected.
The importance of these observations lies in a recent communication
from Pelletier et al.(48) , who maintain that during
DNA-dependent DNA synthesis, the orientation of RT on template-primer
is opposite to that determined by Jacobo-Molina et al.(7) and predicted by Kohlstaedt et al.(6) . According to their model, the RNase H domain
occupies the single-stranded template and not the template-primer
duplex. This predicts that truncating
-helix E` should alter
protection of the single-stranded template rather than the
template-primer duplex. In fact, we observe the opposite. Further proof
that their orientation of RT on template-primer is incorrect lies in
our analysis of replication complexes containing a variant of murine
leukemia virus RT lacking its RNase H domain. While wild type murine
leukemia virus RT protects the template-primer duplex to position
-27, elimination of the RNase H domain has the consequence that
12 base pairs of the template-primer duplex are rendered
DNase-susceptible(39) . These data provide compelling evidence
that during DNA-dependent DNA synthesis, RT is asymmetrically
distributed over the primer 3`-OH with the bulk of contact involving
the template-primer duplex.
Finally, our data illustrate the
importance of qualitative evaluation of HIV RT functions. In the RNase
H assays of Fig. 3, both wild type and p668/p51 RT cleave
the RNA-DNA hybrid with equal efficiency, but differ dramatically in
their final hydrolysis products. When measuring loss of
acid-precipitable counts from a randomly generated RNA-DNA hybrid,
differences between these two enzymes would not be immediately apparent (i.e. loss of counts would be equal). This predicts that drugs
selectively inhibiting directional processing might escape detection,
since quantitative evaluation would indicate no alteration to RNase H
activity. However, we demonstrate here that loss of this function
manifests itself in dramatically reduced DNA strand transfer activity.
Implementing qualitative analysis in drug-screening efforts might then
highlight novel agents which prevent transfer of DNA between RNA
templates during(-)-strand synthesis, exposing stalled
replication intermediates to host nucleases.