Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals*

Matthias GötteDagger , Gottfried Maier, Hans J. Gross§, and Hermann Heumannpar

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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 right-arrow  Gln-PR, respectively (6), as described previously (32). The concentration of RT was determined by A280 measurements using an extinction coefficient of epsilon 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 [gamma -32P]ATP using T4 polynucleotide kinase (Boehringer Mannheim) according to the manufacturer's recommendation. 3'-End labeling with [32P]pCp and T4 RNA ligase (Pharmacia) was carried out as described (36). To ensure homogeneity, labeled nucleic acids were again electrophoretically purified.

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.

To characterize the influence of liganded versus unliganded Fe2+ on the cleavage profile we changed the ratio of free Fe2+ and [Fe(EDTA)]2- by varying the EDTA concentration from 0 to 4 mM while keeping the concentration of all other reaction components constant (Fig. 1). The reaction was allowed to proceed for 5 min at 37 °C and stopped with 40 ml of a solution containing 0.1 M thiourea, 200 ng of tRNA, 10 mM EDTA, and 0.6 M sodium acetate. Samples were subsequently precipitated with ethanol and loaded on a 12% polyacrylamide-urea gel. Products were visualized using a PhosphorImager or by autoradiography overnight.

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 epsilon 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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 1.   [Fe(EDTA)]2--dependent OH-radical footprints of HIV-RT at a DNA template. A, the sequence of the DNA primer/template. The double-stranded DNA primer/template labeled at the 5'-end was incubated with HIV-RT, the primer was elongated by 1 base (ddATP) and the halted complex was subjected to treatment with [Fe(EDTA)]2--dependent OH-radicals, as described under "Experimental Procedures." The protected regions are indicated by bars and the hyperreactive cleavage positions by arrows. B, the cleavage patterns obtained at different ratios of Fe2+ and [Fe(EDTA)]2-. Lanes 1-5 show the [Fe(EDTA)]2--dependent OH-radical footprints obtained at varying EDTA concentrations: without EDTA (lane 1), with 0.4 mM (lane 2), with 0.8 mM (lane 3), with 1.6 mM (lane 4), with 4 mM (lane 5). Lane C shows the pattern without OH-radical treatment and lanes C1 and C2 show the OH-radical patterns of DNA without HIV-RT. The pattern were obtained after electrophoretic separation of the fragments as described under "Methods."

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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Reaction schemes used for generating OH-radicals. A, the modified Fenton reaction. DTT was used to reduce [Fe(EDTA)]1- to regenerate [Fe(EDTA)]2- and maintain the production of OH-radicals (22). B, the potassium peroxonitrite reaction. OH-radicals were generated by disproportioning of the free acid at neutral pH (23).

The role of Fe2+ in the hyperreactive cleavage reaction was analyzed using a different, Fe2+-free OH-radical source for footprinting, namely potassium peroxonitrite (ONOOK) (23-25). The reaction scheme is depicted in Fig. 2B. The free acid has been disproportionated at neutral pH facilitating generation of OH-radicals. Fig. 3 shows a comparison of footprints obtained with ONOOK and [Fe(EDTA)]2-, respectively. The latter was performed at high EDTA concentration to suppress hyperreactivity. The footprints obtained by both methods yield the same protected regions. The lack of enhanced cleavage at the RNase H position in the ONOOK footprint confirms that free Fe2+ is responsible for the enhanced cleavage activity. The slight cleavage enhancement visible in the ONOOK pattern at position -6 might be due to an interaction of the protein with an active intermediate generated when disproportionating ONOOK (25).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of the footprints obtained by [Fe(EDTA)]2- and ONOOK-dependent OH-radicals. The complex of HIV-RT and DNA was formed as described in the legend of Fig. 1. Lane 1 shows the footprint obtained with [Fe(EDTA)]2--dependent OH-radicals using 0.4 mM Fe2+ and 4 mM EDTA, and lane 2 the footprint obtained with ONOOK-dependent OH-radicals as described under "Methods." Lane C shows the pattern without applying OH-radicals and lanes C1 and C2 show the patterns of isolated DNA using [Fe(EDTA)]2--dependent and ONOOK-dependent OH-radicals, respectively.

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+.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 4.   Hyperreactive cleavage at different Mg2+/Fe2+ ratios. The DNA primer/template shown in Fig. 1A was incubated with wild type RT (lanes 1-4) or with the RNase H- mutant (E478Q) (lanes 5-8), as described under "Methods." Hypercleavage was initiated by treatment of the complexes with DTT (5 mM), Fe2+ (0.04 mM), and a varying concentration of Mg2+: without Mg2+ (lanes 1 and 5), with 0.04 mM Mg2+ (lanes 2 and 6), with 0.5 mM Mg2+ (lanes 3 and 7), with 10 mM Mg2+ (lanes 4 and 8). Lane C shows the DNA pattern without HIV-RT and without Fe2+; lane C1 shows the Fe2+-dependent patterns without HIV-RT (0.04 mM Fe2+ and 5 mM 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane), lanes 2 and 3 show the patterns obtained with wild type and mutant HIV-RT, respectively, in the absence of Fe2+.

The hypercleavage reaction in the [Fe(EDTA)]2- assay was dramatically reduced, but not to zero (Fig. 4), when the RNase H- RT mutant (E478Q) (19) was used. The crystal structure model of the RNase H domain (26) indicates that the amino acid replacement in the mutant leads to a reduced affinity of the catalytically active Mg2+. This crystallographic finding along with our result that the same mutation in the RNase H affects the affinity for Fe2+, as well as for Mg2+, suggests that the binding sites for both ions are overlapping, if not identical.

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.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Advance of the hyperreactive cleavage site with progress of DNA synthesis. A, scheme of the stepwise synthesis by HIV-RT on the DNA primer/template. HIV-RT was arrested in the registers 0, 1, 3, 9, and 18 using an incomplete set of nucleotides and a stop nucleotide as described under "Methods." The positions of hypercleavage positions determined from the electrophoretic cleavage pattern in B are indicated by arrows. B, electrophoretic analysis of the hypercleavage products. HIV-RT was incubated with Fe2+ (0.04 mM), DTT (5 mM), DNA primer/template, and the proper nucleotide mixture to obtain complexes arrested in the registers 0 (lane 0), 1 (lane 1), 3 (lane 3), 9 (lane 9), and 18 (lane 18). The size of the cleavage products was determined by using a T-ladder (lane T). Cleavage of DNA without HIV-RT is shown in lane L. Lane C shows the complex without Fe2+ treatment.

The 15-base pair distance previously described by Metzger et al. (18) can be ascribed to an error in their base assignment. In that study higher [Fe(EDTA)]2- concentrations were used, resulting in a larger spread of the cleaved nucleotides (see also Fig. 1B, lanes 1 to 3).

We conclude from the above finding that the Fe2+-dependent hyperreactive cut can be used to trace the position of the Mg2+-binding site of the RT-associated RNase H domain. Mg2+ is replaced by Fe2+, which can generate a high local concentration of OH-radicals, resulting in enhanced cleavage of the nucleotide next to the bound Fe2+. The assumption that hyperreactivity is mediated by an oxidative mechanism is supported by the finding that a reducing agent (DTT) is necessary to maintain cleavage (data not shown). Reduction of Fe2+ is a prerequisite for permanent generation of OH-radicals, as is the case for OH-radical production in solution by the [Fe(EDTA)]2--dependent Fenton reaction (22).

Analysis of the electrophoretic mobility of the 5'-end-labeled cleavage products further confirms an oxidative cleavage mechanism. As depicted in Fig. 5B, cleaved fragments migrate at the same position like the DNA fragments of the T-ladder. The latter was obtained by strand scission with piperidine, which gives rise to 3'-PO4 groups as proposed for OH-radical cleavage (22, 27).

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.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Cleavage and polymerization activity of HIV-RT in the presence of Fe2+. Fe2+-substituted HIV-RT was incubated with primer/templates as indicated and the cleavage products were analyzed by gel electrophoresis as described under "Methods." A, cleavage of an RNA template/primer in register 0 using 5'-end labeled template. A RNA primer/template having the same sequence as that used for DNA cleavage shown in Fig. 1a was used except that the primer was tRNALys-3. The intensity scans were obtained from the electrophoretic cleavage pattern shown as an inset. Lanes 4 (40 µM Fe2+) and 5 (400 µM Fe2+) show the patterns obtained with different Fe2+ concentrations, namely 0.04 mM (lane 4) and 0.4 mM (lane 5). Lane 3 shows the cleavage pattern obtained with Mg2+-substituted HIV-RT. The size of the cleavage products was determined using a G-ladder (lane 1) and an OH ladder (lane 2). B, comparison of the cleavage activity of 3'-labeled RNA template hybridized with RNA or DNA using Fe2+ and Mg2+. Fe2+-substituted HIV-RT was incubated as in the previous experiment using as primer tRNALys-3 or a DNA fragment. The DNA primer has previously been described (9). Its sequence corresponds to the first 24 nucleotides of tRNALys-3. Lanes 1 and 2 show the cleavage pattern obtained by using tRNALys-3 as primer in the presence of 6 mM Mg2+ (lane 1) and 0.04 mM Fe2+ (lane 2); lanes 3 and 4 show the cleavage activities using DNA primers in the presence of Mg2+ (lane 3) and Fe2+ (lane 4). The arrows indicate the cleavage positions. C1 and C2 are control experiments showing that no cleavage occurs in the presence of Fe2+ without HIV-RT if the primer is RNA/RNA (C1) or RNA/DNA (C2). The size of the cleavage products was determined using a G-ladder and an OH- ladder. C, comparison of the polymerization activity of HIV-RT in the presence of Fe2+ and Mg2+. DsDNA using a 5'-labeled primer having the sequence shown in B was incubated with wild type HIV-RT and RNase H- mutant HIV-RT(E478Q), each sample for 1 and 5 min, respectively. The reaction was performed in the presence of 6 mM Mg2+ (left panel) and 0.4 mM Fe2+ (right panel). The efficiency of 3'-end extension was studied by incorporation of ddATP. Lane C is a control experiment where dsDNA was incubated without enzyme, Fe2+ or Mg2+; lanes 1 and 8 are control experiments without enzyme but with Mg2+ and Fe2+, respectively. Incubation without ddATP is shown in lane 2, in the presence of ddATP for 1 min in lane 3 and for 5 min in lane 4. The same reaction was performed with the HIV-RT RNase H- mutant (lanes 5-7). The same set of experiments performed in the presence of Mg2+ was performed in the presence of Fe2+ (lanes 9-14).

Size and nature of the 3'-ends of the cleavage products were characterized by electrophoresis comparing their mobility with that of reference fragments obtained by alkaline hydrolysis of the template. Lane 2 in Fig. 6A shows that the RT-dependent cleavage products migrate between position -18 and -19, indicating a difference of the 3'-ends. Alkaline hydrolysis products contain 3'-PO4 ends, like those generated with RNase T1 (lane 1 in Fig. 6A), thus migrating a little faster than fragments containing 3'-OH ends.

We conclude from the above findings: first, that the RNase H activity of HIV-1 RT cleaves the RNA template at position -18 with Fe2+ as well as with Mg2+ leaving a 3'-OH group, and second, that Fe2+ can replace the catalytically active Mg2+ at the RNase H active site without affecting the enzymatic cleavage mechanism. In other words, the absence of cleavage products containing 3'-PO4 ends suggests that there is no Fe2+-dependent cleavage due to site-specific generated hydroxyl radicals when dsRNA is used as primer/template. The minor cleavage product at position -17 appearing in the Fe2+-dependent assays (lanes 4 and 5 of Fig. 6A) might be attributable to a 5'-directed cleavage activity of HIV-RT due to a polymerase-independent cleavage mechanism previously described for the Mg2+-dependent assay (6, 7, 9-12).

To visualize Fe2+-dependent cleavages on a DNA/RNA substrate, we have used a 3'-end-labeled RNA template. This allowed us to determine specifically the polymerase-dependent RNase H cut (9), that reflects the binding mode in which RT's polymerase active site is located in the vicinity of the primer 3'-end. Cleavage positions were compared on dsRNA and DNA/RNA hybrids using Mg2+ and Fe2+, respectively. The electrophoretic pattern shown in Fig. 6B indicates that the main cleavage site at the RNA template is located 18 nucleotides upstream of the primer 3'-end, regardless of whether the primer is RNA (lanes 1 and 2) or DNA (lanes 3 and 4). Additionally, the cleavage position is also identical in regard to the type of the metal ion used, although the efficiency of the cleavages is modulated. Fe2+-dependent cuts are weaker than their Mg2+-dependent counterparts. If the primer is tRNALys-3 (lanes 1 and 2), a single cleavage site is observed. This is in line with previous studies which showed that dsRNA is cleaved in the polymerization-dependent mode 18 bases upstream of the 3'-primer position (9). If the primer is a DNA fragment (lanes 3 and 4), more than one cleavage is observed; this is probably due to cleavage of the template in a polymerization-independent mode indicated by small arrows in Fig. 6B.

To elucidate whether Fe2+ may also serve as a catalyzing ion in regard to RT's polymerase activity, we have followed the extension of a 5'-end-labeled DNA primer with ddATP using the DNA/DNA substrate depicted in Fig. 1A. Fig. 6C shows that Fe2+ can in fact substitute Mg2+ without largely affecting DNA synthesis of the wild-type enzyme (lanes 3 and 4 compared with 10 and 11) and the RNase H-deficient enzyme (lanes 6 and 7 compared with 13 and 14), respectively. Thus, Fe2+ catalyzes cleavage and polymerase activities, although less efficiently compared with the Mg2+-dependent reactions.

It is worth noting that the Fe2+ concentrations chosen in this experiment is a compromise to fulfill two requirements. The concentration must be sufficiently high to facilitate the Fe2+-dependent enzymatic functions, but below 0.4 mM to avoid attack of DNA or RNA by OH-radicals generated by Fe2+ free in solution.

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).


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 7.   Fe2+-dependent hydroxyl radical cleavage of HIV-1 RT. A, cleavage of HIV-RT, isolated and in the complex with primer/template. HIV-RT, wild type (left panel, lanes 2-4), and RNase H- mutant (E478Q) (right panel, lanes 6-8), were incubated with dsRNA primer/template (lanes 4 and 8), with dsDNA primer/template (lanes 3 and 7), and without primer/template (lanes 2 and 6), treated by Fe2+-dependent hydroxyl radicals, as described under "Experimental Procedures," and subjected to SDS-PAGE. Lanes 1 and 5 show the wild type and RNase H--mutant, respectively, without OH radical treatment, as a reference. The subunits and the cleavage products were stained by Coomassie. B, competition experiment with Mg2+. The Fe2+-substituted HIV-RT has been incubated with dsDNA primer/template as shown in Fig. 1A and has been treated with increasing amounts of Mg2+ (c = 0 mM, lane 3; c = 0.4 mM, lane 4; c = 1 mM, lane 5; c = 5 mM, lane 6; c = 10 mM, lane 7; c = 20 mM, lane 8. Lane 1 shows the HIV-RT without Fe2+ and lane 2 shows HIV-RT treated with Fe2+ (c = 0.4 mM) but without primer/template.

The molecular weight of the cleavage products is in the range of 51 to 66 kDa. The size of the cleavage products indicates that only the p66 subunit is cleaved. This is in line with the observation on dsDNA that OH-radicals are generated solely by Fe2+ bound at the catalytic site of the RNase H site in p66. The cleavage efficiency is dramatically reduced (Fig. 7A, right panel) if the RNase H- mutant HIV-RT is used, indicating that intactness of the RNase H site is required for generation of OH-radicals by Fe2+, which is in accord with the conclusion drawn from dsDNA cleavage experiments by Fe2+-substituted HIV-RT.

The protein cleavage is much less efficient than nucleic acid cleavage, as a comparison of the cleavage patterns in Fig. 7 and Fig. 1A shows. Protein is obviously less susceptible to OH-radical attack than nucleic acid.

HIV-RT is cleaved by OH-radicals to the same extent, regardless of whether dsRNA (Fig. 7A, lane 3) or dsDNA (lane 4) is used. This excludes the possibility mentioned above, that the lack of OH-radical cleavage in dsRNA can be explained by a reduced OH-radical production with dsRNA as primer/template.

The cleavage patterns obtained with dsRNA (Fig. 7A, lane 3) and dsDNA (lane 4) are identical. Two cleavage products are observed having a molecular mass of 55 and 60 kDa. The fact that the cleavage patterns with dsDNA and dsRNA are the same suggests that the conformation of HIV-RT at the Fe2+-binding site does not change, regardless of whether the primer/template is dsRNA or dsDNA.

It is interesting to note that the cleavage pattern differs depending on whether HIV-RT is isolated or bound to a nucleic acid primer/template. The OH-radical pattern of isolated HIV-RT shows in Fig. 7A, lane 2, an additional band having a molecular mass of about 57 kDa, indicating a conformational difference of the isolated and complexed HIV-RT around the Fe2+/Mg2+-binding site of the RNase H domain.

If the postulated replacement mechanism of Fe2+ and Mg2+ as observed with template cleavage is correct, it should be also possible to suppress the Fe2+-dependent OH-radical cleavage of the protein by excess of Mg2+. Fig. 7B shows that the cleavage of the p66 subunit is fully suppressed in the presence of 20 mM Mg2+ (lane 8), indicating that the OH-radical generating Fe2+ can be replaced by Mg2+.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

par 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Skalka, A.-M., and Goff, S. P. (eds) (1993) Reverse Transcriptase, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  2. Litvak, S., Sarih-Cottin, L., Fournier, M., Andreola, M., and Tarrago-Litvak, L. (1993) Trends Biochem. Sci. 19, 114-118
  3. Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B., and Kleiman, L. (1993) J. Virol. 67, 3246-3253[Abstract]
  4. Isel, C., Lanchy, J. M., Le Grice, S. F., Ehresmann, C., Ehresmann, B., and Marquet, R. (1996) EMBO J. 15, 917-924[Abstract]
  5. Wöhrl, B. M., and Moelling, K. (1990) Biochemistry 29, 10141-10147[Medline] [Order article via Infotrieve]
  6. Schatz, O., Mous, J., and Le Grice, S. F. J. (1990) EMBO J. 9, 1171-1176[Abstract]
  7. Gopalakrishnan, V., Peliska, J. A., and Benkovic, S. J. (1992) Proc. Natl. Acad. Sci. U. S. A.. 89, 10763-10767[Abstract]
  8. Peliska, J. A., and Benkovic, S. J. (1992) Science 258, 1112-1118[Medline] [Order article via Infotrieve]
  9. Götte, M., Fackler, S., Hermann, T., Perola, E., Cellai, L., Gross, H. J., Le Grice, S. F. J., and Heumann, H. (1995) EMBO J. 14, 833-841[Abstract]
  10. Furfine, E. S., and Reardon, J. E. (1991) J. Biol. Chem. 266, 406-412[Abstract/Free Full Text]
  11. DeStefano, J. J., Buiser, R. G., Mallaber, L. M., Myers, T. W., Bambara, R. A., and Fay, P. J. (1991) J. Biol. Chem. 266, 7423-7431[Abstract/Free Full Text]
  12. DeStefano, J. J., Buiser, R. G., Mallaber, L. M., Bambara, R. A., and Fay, P. J. (1991) J. Biol. Chem. 266, 24295-24301[Abstract/Free Full Text]
  13. DeStefano, J. J., Mallaber, L. M., Fay, P. J., and Bambara, R. A. (1994) Nucleic Acids Res. 22, 3793-3800[Abstract]
  14. Kati, W. M., Johnson, K. A., Jerva, L. F., and Anderson, K. S. (1992) J. Biol. Chem. 267, 25988-25997[Abstract/Free Full Text]
  15. Ben-Artzi, H., Zeelon, E., Le Grice, S. F. J., Gorecki, M., and Panet, A. (1992) Nucleic Acids Res. 89, 927-931
  16. Hostomsky, Z., Hughes, S. H., Goff, S. P., and Le Grice, S. F. J. (1994) J. Virol. 68, 1970-1971[Abstract]
  17. Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324[Abstract]
  18. Metzger, W., Hermann, T., Schatz, O., Le Grice, S. F. J., and Heumann, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5909-5913[Abstract]
  19. Schatz, O., Cromme, F. V., Grüninger-Leitch, F., and Le Grice, S. F. J. (1989) FEBS Lett. 257, 311-314[CrossRef][Medline] [Order article via Infotrieve]
  20. Ettner, N., Metzger, J. W., Lederer, T., Hulmes, J. D., Kisker, C., Hinrichs, W., Ellestad, G. A., and Hillen, W. (1995) Biochemistry 34, 22-31[Medline] [Order article via Infotrieve]
  21. Zaychikov, E., Martin, E., Denissova, L., Kozlov, M., Markotsov, V., Kashlev, M., Heumann, H., Nikiforov, V., Goldfarb, A., and Mustaev, A. (1996) Science 273, 107-109[Abstract]
  22. Tullius, T., and Dombroski, B. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5469-5473[Abstract]
  23. King, P. A., Anderson, V. E., Edwards, J. O., Gustafson, G., Plumb, R. C., and Suggs, J. W. (1992) J. Am. Chem. Soc. 114, 5430-5432
  24. King, P. A., Jamison, E., Strahs, D., Anderson, V. E., and Brenowitz, M. (1993) Nucleic Acids Res. 21, 2473-2478[Abstract]
  25. Götte, M., Marquet, R., Isel, C., Anderson, V. E., Keith, G., Gross, H. J., Ehresmann, C., Ehresmann, B., and Heumann, H. (1996) FEBS Lett. 390, 226-228[CrossRef][Medline] [Order article via Infotrieve]
  26. Davies, J. F., II, Hostomska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 252, 88-95[Medline] [Order article via Infotrieve]
  27. Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564[Abstract]
  28. Sigman, D. S. (1990) Biochemistry 29, 9097-9105[Medline] [Order article via Infotrieve]
  29. Pogozelski, W. K., McNeese, T. J., and Tullius, T. D. (1995) J. Am. Chem. Soc. 117, 6428-6433
  30. Pelletier, H., Sawaya, M. R., Kumar, R., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903[Medline] [Order article via Infotrieve]
  31. Wöhrl, B. A., Georgiadis, M. M., Telesnitsky, A., Hendrickson, W. A., and Le Grice, S. F. J. (1995) Science 267, 96-99[Medline] [Order article via Infotrieve]
  32. Le Grice, S. F. J., and Grüninger-Leitch, F. (1990) Eur. J. Biochem. 178, 307-314
  33. Raba, M., Limburg, K., Burghagen, M., Katze, J. R., Simsek, M., Heckmann, J. E., RajBhandary, U. L., and Gross, H. J. (1979) Eur. J. Biochem. 97, 305-318[Abstract]
  34. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  35. Saenger, W. (1983) Principles of Nucleic Acid Structure, pp. 220-282, Springer Verlag, New York
  36. Bruce, A. G., and Uhlenbeck, O. C. (1978) Nucleic Acids Res. 5, 3665-3677[Abstract]
  37. Fedoroff, O. Y., Salazar, M., and Reid, B. R. (1993) J. Mol. Biol. 233, 509-523[CrossRef][Medline] [Order article via Infotrieve]
  38. Fedoroff, O. Y., Ge, Y., and Reid, B. R. (1997) J. Mol. Biol. 269, 225-239[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.