From the Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany and the § Bayerische Julius-Maximilians-Universität, Institut für Biochemie, Biozentrum, Am Hubland, D-97074 Würzburg, Germany
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ABSTRACT |
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Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.
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INTRODUCTION |
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Retroviral reverse transcriptases (RTs)1 are multifunctional enzymes having an RNA-dependent and a DNA-dependent polymerase activity and a ribonuclease H (RNase H) activity, which degrades the RNA strand of RNA/DNA hybrids (1). These activities facilitate the conversion of single-stranded genomic RNA to the double-stranded proviral DNA which is integrated into the host genome. Synthesis of the first DNA strand, the minus strand DNA, is initiated from a cellular tRNA which binds with its 3' terminus to the complementary primer-binding site near the 5'-end of the viral RNA (2-4). The newly synthesized minus-strand DNA serves in turn as template for the synthesis of the second DNA strand or plus-strand. Prerequisite for the plus-strand synthesis is removal of the viral RNA from the minus-strand DNA. Both activities act simultaneously, presupposing a spatial and temporal interdependence of the active sites.
The interplay of the two activities has been studied extensively in RT of human immunodeficiency virus type 1 (HIV-1) (5-13). It has been demonstrated that the viral RNA template is cleaved 18 nucleotides upstream with respect to the 3'-end of the nascent primer terminus (7) during minus-strand synthesis. But cleavage does not occur after each nucleotide incorporation step, indicating that the two activities, the polymerase and RNase H activity, are not coupled in a 1:1 mode (11-14). Gopalakrishnan et al. (7) suggested a temporal coordination of the two activities due to kinetic coupling. Cleavage can occur if the rate of DNA polymerization is lower than the rate of RNA hydrolysis. A similar concept might also explain the resistance of dsRNA toward cleavage. It has been shown that dsRNA formed by the tRNALys-3 and the viral RNA during initiation of minus-strand synthesis remains intact during DNA synthesis, but is cleaved, if the DNA synthesis is artificially stopped through use of chain terminating nucleotides (9). This can be explained by the fact that the rate of polymerization is higher than the rate of RNA template hydrolysis of dsRNA in line with the kinetic coupling model (9). Ribonuclease activity on dsRNA, which is believed to be mediated by the active site of the RNase H domain (9, 15), has been termed RNase H* activity (16).
The high resolution crystallographic model of an RT-dsDNA primer/template complex has revealed that the space between the polymerization site and the RNase H site can be filled by a primer/template of 17 or 18 bases (17).
In the present study a biochemical approach has been applied, namely
site-specific hydroxyl radical cleavage to localize the active site of
HIV-1 RT-associated RNase H domain on a DNA template. This approach is
based on findings by Metzger et al. (18) who showed that a
hypersensitive cleavage site appears in the [Fe(EDTA)]2
footprint close to the position where the RNase H active site is
located at the template. This cleavage reaction requires an intact
RNase H domain, since hyperreactivity was not observed with an RNase
H-deficient enzyme (HIV-1 RT(E478Q)), whose ability to bind divalent
metal ions is supposed to be suppressed (19). Hyperreactivity was
previously interpreted as an enhanced accessibility of the template due
to a putative conformational change of the DNA induced by the RNase H
domain (18). This report shows that hyperreactivity is due to
site-specific generated hydroxyl radicals released by
Fe2+-ions after replacement of Mg2+ at the
RNase H active site.
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EXPERIMENTAL PROCEDURES |
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Materials
Preparation of HIV-RT--
Heterodimeric HIV-1 RT (p66/p51) and
the RNase H-deficient mutant enzyme (p66E478Q/p51) were
prepared by metal chelate and ion exchange chromatography from
Escherichia coli strains
M15::pDM1.1::pRT6H-PR (32) and M15::pDM1.1::pRT6HGlu Gln-PR,
respectively (6), as described previously (32). The concentration of RT
was determined by A280 measurements using an
extinction coefficient of
280 nm = 1.4 mg/ml
cm
1. RT preparations were stored at
20 °C in 50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 1 mM
1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, and 50%
glycerol. Since glycerol scavenges hydroxyl radicals, RT preparations
were dialyzed for 1 h against 50 mM Tris-HCl, pH 7.8, 50 mM NaCl before incubating with the primer/template substrate (see below).
Nucleic Acids-- DNA was chemically synthesized using the phosphoamidite method. To remove impurities, oligonucleotides were electrophoretically purified using 12% polyacrylamide, 7 M urea gels containing 50 mM Tris borate, pH 8.0, 1 mM EDTA. DNA was visualized by UV shadowing at 254 nm and the fragment of correct length was eluted from excised gel slices with 0.5 M ammonium acetate, pH 6.5, 0.1% SDS. After ethanol precipitation, concentration of nucleic acids was determined spectrophotometrically.
RNA (designated as primer-binding site 1 by Götte et al. (9) was synthesized by in vitro transcription using T7 RNA polymerase. Purification of the transcript was achieved as described above. tRNALys-3 with the anticodon SUU (S = 5-methoxycarbonyl-2-thiouridine) was isolated from rabbit liver and purified following the procedure from Raba et al. (33). End labeling of dephosphorylated RNA transcript and the DNA template was conducted with [Methods
Formation of RT-Primer-Template Complexes and Polymerization-- To achieve quantitative complex formation, primer/template sequences were hybridized before incubating with the enzyme. Annealing was conducted in a reaction volume of 10 ml. A mixture of 100 nM (final concentration) unlabeled DNA template, 50,000 cpm of 5'-end-labeled template, and 150 nM DNA primer in a buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM NaCl was heated to 90 °C for 1 min followed by incubation at 60 °C for 10 min and cooling for 20 min to room temperature. Complete hybridization was confirmed by analyzing aliquots of the annealed duplexes on native polyacrylamide gels containing 20 mM Tris-HCl, pH 7.8, 10 mM NaCl. The tRNALys-3/RNA homoduplex and the DNA/RNA heteroduplex were prepared analogously.
In a reaction volume of 20 ml, pre-annealed primer/template substrates (100 nM) were incubated for 5 min at 37 °C with HIV-RT (150 nM) or the RNase H-deficient enzyme, respectively, in a buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM NaCl. Polymerization was then initiated by addition of MgCl2 (6 mM) and the appropriate dNTP (100 mM)/ddNTP (500 mM) mixture permitting primer extension by 1, 3, 9, and 18 nucleotides. Under these reaction conditions 95% of the primer is correctly elongated after 10 min. Fe2+-dependent incorporation of ddATP was initiated by addition of 0.4 mM Fe2+.[Fe(EDTA)]2-dependent OH-radical
Footprinting--
The standard hydroxyl radical footprinting using the
modified Fenton reaction was performed essentially as recently
described (22, 25). Pre-formed RT-primer-template complexes were
incubated in a total reaction volume of 25 ml with a mixture of
Fe(NH4)SO4·6H2O (400 µM), EDTA (800 µM), 5 mM DTT,
and 0.05% H2O2.
ONOOK-dependent OH-radical Footprinting--
A
stable alkaline solution of ONOOK, pH 12.5, was synthesized as
described (24). The ONOOK concentration was determined as 60 mM by absorbance measurement at 302 nm using an extinction coefficient of 302 nm = 1670 M
1 cm
1. The peroxonitrite
solution was stored at
80 °C and thawed prior to use. Cleavage
reactions with ONOOK were conducted by adding 1 ml of the peroxonitrite
solution to the sample solution buffered at pH 7 (24, 25).
RT-primer-template complexes were prepared in a buffer containing 80 mM sodium kakodylate, pH 7, and 20 mM NaCl.
Reactions were stopped and analyzed essentially as described above.
Fe2+/Mg2+ Competition Experiment-- Pre-formed RT-primer-template complexes in a buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM NaCl, and 6 mM MgCl2 were incubated in a total reaction volume of 25 µl with a mixture of Fe(NH4)SO4·6H2O (40 µM) and 5 mM DTT. The reaction proceeds for 10 min at 37 °C and was stopped as described in the previous paragraph. Replacement of Mg2+ at the active site of RT-associated RNase H domain by Fe2+ was studied by applying MgCl2 in a concentration range from 0 to 10 mM and by keeping the Fe(NH4)SO4·6H2O concentration constant. The size of the Fe2+-dependent cleavage products was assigned by a T-ladder generated after modifying the DNA with OsO4/bipyridine followed by cleaving the sugar-phosphate backbone with piperidine.
RNase H/H* Activities in the Presence of Fe2+ and Mg2+-- RNase H* activity was monitored on the preformed RT-tRNALys-3·RNA complex in the presence of 6 mM MgCl2 (9). RNase H activity was monitored analogously using the heteroduplex shown in Fig. 6B. To characterize cleavage specificity with Fe2+ the complex was incubated in the absence of MgCl2 using various concentrations of Fe(NH4)SO4·6H2O (40-400 µM). The band intensities of the cleavage products did not depend on the presence of a reducing agent, such as DTT (data not shown). RNA fragment sizes were assigned using a homogeneous RNA ladder generated by partial alkaline hydrolysis with Na2CO3/NaHCO3, pH 9.5, and a G-ladder obtained by partial digestion of the RNA with ribonuclease T1.
Fe2+-dependent Hydroxyl Radical Cleavage of HIV-1 RT-- To analyze the Fe2+-dependent hydroxyl radical cleavage products in the protein HIV-RT, dsRNA and dsDNA primer/template (concentration = 100 nM) was incubated with equal amounts of RT (the wild type and the RNase H-deficient enzyme, respectively). In a reaction volume of 20 µl, RT and primer/template were incubated with ddATP permitting elongation of the primer by one nucleotide. The complex was subjected to Fe2+ treatment (0.4 mM Fe(NH4)SO4·6H2O) at 37 °C for 10 min in the presence of 5 mM DTT. The reactions were stopped by adding 1 volume of SDS-PAGE application buffer. The reaction mixture was heated for 5 min at 95 °C and loaded on a 12% SDS-PAGE (34). The gel was stained with Coomassie Brilliant Blue R-250.
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RESULTS |
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To compare the hyperreactive cleavages on a DNA template with the
RNase H-induced cuts recently obtained on an RNA template (9), we used
a dsDNA fragment having the same sequence (Fig. 1A). Complexes of
primer/template DNA and HIV-RT were formed and subjected to
[Fe(EDTA)]2-dependent OH-radical treatment,
as described under "Methods." Lane 3 in Fig.
1B shows the OH-radical footprint of HIV-1 RT that was
obtained using 5'-end-labeled DNA template. The schematic representation of the footprint in Fig. 1A indicates the
contacts between HIV-RT and template. The region covered by HIV-RT
reaches from base position +4 to
18 containing a window of
accessibility around base position
10 and a region of hyperreactive
cleavage at base positions
15 to
18. This pattern essentially
agrees with that previously published by Metzger et al.
(18). However, that the observed hyperreactivity reflects an enhanced
accessibility of the template DNA due to an RNase H-induced distortion
of the template does not seem to be convincing. An accessibility which exceeds that of free DNA is difficult to envision. Therefore, we
investigated whether the hypercleavage could be due to another reason.
It was previously shown with other Mg2+-binding proteins
such as tetracycline repressor (20) and E. coli RNA
polymerase (21) that Fe2+ can replace Mg2+ and
generate site-specific OH-radicals which cleave the DNA. Whether this
mechanism is also applicable to the catalytic Mg2+ of the
RT-associated RNase H, is discussed below.
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Fe2+ Is Responsible for Hyperreactive Cleavage--
In
a typical OH-radical footprinting experiment, the Fenton reaction is
used for generating OH-radicals which cleave the DNA (Fig.
2A). The OH-radicals most
likely attack the nucleotide sugars at the C-1 and C-4 positions,
leading to excision of the base. EDTA is used in a 2-fold excess over
Fe2+. Thus the negatively charged
[Fe(EDTA)]2 complex is obtained which prevents
interaction of iron with the sugar phosphate backbone of the DNA (22).
The Fe2+-dependent OH-radical footprint then
reflects the accessibility of DNA toward diffusable OH-radicals
generated by the metal complex. Hyperreactive cleavage is observed only
when Fe2+ is in excess. The role of excess Fe2+
was analyzed by shifting the equilibrium between Fe2+ and
[Fe(EDTA)]2
in the Fenton reaction. This was achieved
by varying the EDTA concentration (see Fig. 2A). Fig.
1A depicts the footprints of HIV-RT obtained at different
EDTA concentrations. The intensity of the hypersensitive cuts centered
at base position
16 decreases in step with increasing EDTA
concentration. At a 10-fold molar excess of EDTA over Fe2+
(lane 5 in Fig. 1B) no hypercleavage is observed.
Comparison of the footprints in lane 5 with those in
lanes 1-4 shows that hypercleavage occurs within
the fully protected region. The strongest cuts are seen in the absence
of EDTA (lane 1 in Fig. 1B). This result shows
that hyperreactivity is attributable to Fe2+, and not to
the [Fe(EDTA)]2
complex.
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Fe2+ Displaces Mg2+ at the RNase H Active
Site--
To prove that hypercleavage is due to a replacement of
Mg2+, a competition experiment using wild type RT as well
as the RNase H-deficient RT(E478Q) mutant enzyme was performed as
described under "Methods." Fe2+ was kept constant and
the Mg2+ concentration was varied. To obtain cleavage at a
single position the standard conditions were slightly changed. EDTA was
omitted and the concentration of Fe2+ was reduced from 400 to 40 µM. Due to these changes only hypercleavage at
position 17 and no OH-radical footprint was observed in contrast to
the previously shown Fe2+ assays (Figs. 1 and 3). The
result of the Fe2+/Mg2+ competition experiment
is shown in Fig. 4. The yield of
hypercleavage decreases with increasing Mg2+ concentration
indicating that Mg2+ displaces Fe2+.
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The DNA Template Is Cleaved 17 Nucleotides Upstream of the Primer 3'-End-- To determine the exact position of the hypersensitive cut, HIV-1 RT was arrested at different positions during synthesis by using an incomplete set of dNTPs along with a chain terminating ddNTP (Ref. 18, "Experimental Procedures"). After the primer was specifically extended by 1, 3, 9, and 18 nucleotides (Fig. 5A), complexes were subjected to treatment with Fe2+-generated OH-radicals. Determination of the cleavage position was facilitated by lowering the concentration of Fe2+ so that only a single nucleotide was cleaved. The cleavage positions were assigned by means of a T-ladder. Thymidines of the single-stranded template strand were modified by OsO4-bipyridine, followed by strand scission with piperidine, as described under "Experimental Procedures." The pattern in Fig. 5B and the schematic representation in Fig. 5A shows that the template DNA strand is cleaved 17 base positions upstream of the primer 3'-end in all registers, indicating that the 17-base distance is sequence-independent.
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Comparison of Cleavage Positions on RNA and DNA Templates-- The observation that the Fe2+-dependent cut in different registers occurs at a constant distance from the primer 3'-end is reminiscent of the RNase H*-induced cuts obtained when analyzing complexes stalled on dsRNA during initiation of minus-strand synthesis (9). These studies showed that the RNA template of the tRNALys-3/primer-binding site duplex was cleaved in the presence of Mg2+ 18-base positions upstream of the primer 3' terminus, while DNA used in the present study was cleaved by the Fe2+-substituted HIV-RT 17-base positions upstream.
Whether the cleavage position depends on the type of metal ion used was analyzed by cleavage studies using the same RNA primer/template, tRNALys-3/primer-binding site, in the presence of Mg2+ as well as of Fe2+. Cleavage was performed using wild type RT. The conditions were the same as described in the previous experiment. The cleavage products were analyzed by means of a 5' label in the template RNA which provides information about the chemical nature of the 3' terminus by gel electrophoretic mobility studies, as described above. The obtained cleavage patterns are depicted in Fig. 6A. Comparison of lane 3 with lanes 4 and 5 in Fig. 6A shows that the electrophoretic mobility of the major cleavage products obtained in the presence of Fe2+ and Mg2+ are the same, indicating that cleavage occurred at the same base position and that the 3'-ends are identical.
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Fe2+-dependent OH-radical Cleavage of HIV-RT-- The cleavage pattern does not differ if HIV-RT is bound to dsRNA or dsDNA but changes if HIV-RT is analyzed without nucleic acid primer/template. It is surprising that no OH-radical cleavage products are observed with dsRNA as a primer/template in contrast to dsDNA. To exclude the possibility that the lack of oxidative cleavage products in dsRNA is due to a reduced OH-radical production, we analyzed whether and how Fe2+-dependent OH-radicals cleave also the protein, the HIV-RT.
HIV-RT, wild type, and RNase H-mutant HIV-RT(E478Q), was incubated with dsDNA, dsRNA and without primer/template in the presence of Fe2+. The incubation and cleavage conditions were the same as in the experiment described in Fig. 1 for nucleic acid cleavage. SDS-PAGE (Fig. 7) was used to analyze the OH-radical cleavage on HIV-RT. Cleavage products are observed in isolated HIV-RT (Fig. 7A, lane 2) and in the complex with dsRNA (lane 4) and dsDNA (lane 3).
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DISCUSSION |
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The [Fe(EDTA)]2-dependent OH-radical
footprint of HIV-RT on dsDNA shows hyperreactive cuts (18) at the
upstream edge of the footprint. To determine whether this cut can be
used to trace the position of the active center of RT-associated RNase
H, we have analyzed the mechanism of the hypercleavage reaction. We show that the template DNA is cleaved by OH-radicals which are generated at the metal-binding site of the RNase H active site after
replacement of Mg2+ by Fe2+.
The conclusion that unliganded Fe2+ is responsible for the
hypercleavage is based on the finding that the hyperreactive cleavage is observed only in the
[Fe(EDTA)]2-dependent OH-radical footprint
and not in the Fe2+ free (ONOOK)-dependent
OH-radical footprint; the intensity of the hyperreactive cleavage
depends on the availability of free Fe2+ in the Fenton
reaction.
The conclusion that the hyperreactive cleavage is facilitated by
OH-radicals is supported by the finding that cuts are spread over a
region of four to five nucleotides centered around the main cleavage
site at position 17, in line with the assumption that OH-radicals
diffuse from the Fe2+ source and cleave the most proximate
base most efficiently; analysis of the cleavage product, which shows
that the 3'-end contains PO4 group as expected for hydroxyl
radical mediated cuts (28, 29). That a reducing agent such as DTT is
required further supports the view that hyperreactivity is mediated by
an oxidative process (data not shown).
The conclusion that Fe2+ binds at the metal-binding site of
RT-associated RNase H active site is based on the findings that hyperreactive cleavage is not observed with the RNase H
mutant (E478Q) in which the Mg2+-binding site is destroyed
by replacement of glutamic acid 478 by glutamine (6); that the
intensity of the hyperreactive cleavage can be modulated by the
Fe2+/Mg2+ ratio in the assay. Excess
Mg2+ reduces the hypercleavage, indicating that
Fe2+ and Mg2+ probably compete for the same
site. But it is not clear whether one or both of the two
Mg2+ ions evidenced by the crystal structure of RNase H
(26) can be replaced by Fe2+.
Mg2+ ions are essential not only for the function of the RT-associated RNase H active site but also for the DNA polymerization site. In contrast to other Mg2+-dependent polymerases, such as E. coli RNA polymerase (21), HIV-RT shows no hypercleavage of the template at the polymerization site after replacement of Mg2+ by Fe2+. That Mg2+ is replaced in HIV-RT also at the DNA polymerization active site is clear from DNA polymerization studies in the presence of Fe2+. DNA polymerization takes place, but with lower efficiency, if 0.4 mM Fe2+ is used instead of the standard 6 mM Mg2+ (Fig. 6C).
There is no obvious reason for the lack of hyperreactive cleavage of the template at the polymerization site by OH-radicals. It might be that Fe2+ at the polymerization site has a lower redox potential or that the accessibility of Fe2+ by oxygen or diffusion of H2O2 is reduced due to sterical reasons.
Our studies show that hypercleavage on DNA can be spatially and mechanistically linked with the Fe2+ substitution at the metal-binding site of the RT-associated RNase H active site. This permits conclusions to be drawn about the orientation of HIV-RT on a dsDNA. The orientation has been controversially discussed, since the crystal structure of rat DNA polymerase b with a DNA primer/template and ddCTP showed that RT binds its dsDNA substrate in the opposite orientation (30). In this binding mode, the RNase H domain would contact the single-stranded template downstream of the primer terminus. Our hypercleavage study excludes such a binding mode for HIV-RT in line with previous results from differential DNase I footprinting studies using murine leukemia virus RT with and without RNase H domain by Wöhrl et al. (31).
HIV-RT appears rather flexible with respect to the requirements of the type of metal ions catalyzing the cleavage reaction on RNA. Mg2+ as well as Fe2+ is accepted. The cleavage mechanism is an enzymatic mechanism for both metal ions, as indicated by the analysis of the 3' termini of the cleavage products which is a 3'-OH (Fig. 6A in this study and Götte et al. (9)). It is not quite clear why no oxidative cleavage products by OH-radicals are observed with the Fe2+-substituted HIV-RT with RNA. We can exclude the possibility that no OH-radicals are generated if dsRNA is the template, since protein cleavage is observed regardless of whether the template/primer is dsRNA or dsDNA. The reason for the absence of oxidative cleavage products could be due to greater efficiency of the enzymatic cleavage process and/or due to the fact that OH-radicals cleave at the same position.
The cleavage on dsDNA and dsRNA differs with respect to both position and mechanism, although the same catalyzing ion, namely Fe2+, was used for both primer/templates. It is clear that dsDNA can only be cleaved by an oxidative process, since enzymatic cleavage is not possible on DNA. DsRNA is cleaved essentially by an enzymatic mechanism, although OH-radicals are generated. This can be explained by the fact that the oxidative cleavage process is less efficient than the enzymatic process.
The most interesting question is why DNA and RNA are cleaved at different positions. While the template strand of dsDNA is cleaved 17 base positions upstream of the 3'-primer terminus, dsRNA and DNA/RNA is cleaved 18 base positions upstream.
It is unlikely that the difference in the position can be attributed to difference in the cleavage mechanisms for reasons pointed out above or due to a conformational difference of HIV-RT when bound to dsDNA, dsRNA, or DNA/RNA. The latter is supported by the finding that the OH-radical cleavage pattern of HIV-RT is the same with dsDNA and dsRNA. The most likely explanation for the difference in the cleavage position is a conformational difference of the RNA and the DNA primer/template in the complex with HIV-RT. The high resolution structure model of HIV-RT in complex with a dsDNA fragment (17) shows that the template/primer consists of two segments, a 7-base pair region in A-type conformation located at the 3'-primer terminus and a 11-base pair region in B-type conformation further upstream having a kink in between. It was suggested that the reason for the B-like conformation of the upstream located in the 11-base pair segment is its enhanced solution accessibility and the reason for the A-like conformation of the downstream located in the 7-base pair region is its strong interaction with HIV-RT (17). Based on the data of our study we suggest that dsRNA and DNA/RNA have an A-like conformation not only in the 7-base pair downstream region but also in the 11-base pair upstream region. This suggestion is supported by the postulated solution accessibility of the upstream region which would mean that dsRNA and DNA/RNA adopts in this region the canonical solution structure which is the A-conformation (35, 37, 38). Since A-RNA has a lower pitch than B-DNA (about 30 Å and 34 Å, respectively (35), it permits the accommodation of one additional base pair within a helical turn. This interpretation is in line with our observation that dsDNA primer/template is cleaved 1-base position closer to the 3'-end than dsRNA and DNA/RNA.
Therefore, we suggest that differences in the cleavage pattern obtained by Fe2+-dependent enzymatic and oxidative cleavage of RNA and DNA can be used to trace differences of the helical pitch of the primer/template region between the polymerization site and the RNase H site.
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ACKNOWLEDGEMENTS |
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We thank Stuart F. J. Le Grice for
providing the strains overexpressing HIV RT and the RNase
H mutant, Mark A. Wainberg for valuable discussions, and
Vernon E. Anderson for providing ONOOK.
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FOOTNOTES |
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* This work was supported in part by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: McGill University AIDS Centre, Lady Davis
Institute-Jewish General Hospital, Montréal, Québec H3T
1E2, Canada.
¶ Supported by SFB 165 and Fonds der chemischen Industrie.
To whom correspondence should be addressed.
1 The abbreviations used are: RT, reverse transcriptase; HIV-1, human immunodeficiency virus type 1; RNase H, ribonuclease H; dNTP, 3'-deoxynucleoside triphosphate; ddNTP, 2',3'-dideoxynucleoside triphosphate; OH-radicals, hydroxyl radicals; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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