Department of Material and Life Science, Graduate School of Engineering, Osaka University, 21, Yamadaoka, Suita, Osaka 565-0871, Japan
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Abstract |
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Keywords: cross-linking/polypurine tract/ribonuclease H/sequence specificity/Thermus thermophilus
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Introduction |
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The specificity and catalytic efficiency of DNA-linked RNase H have been shown to be affected by the size of the linker between the enzyme and DNA (Uchiyama et al., 1994) and the size of DNA adduct (Kanaya et al., 1994
). Of the various DNA-linked RNases H with different linkers in size, ranging from 18 to 27 Å, that with a 27 Å linker cleaved an oligomeric RNA substrate with the highest efficiency and site-selectivity. Likewise, of the various DNA-linked RNases H with different DNA adducts in size, ranging from 7- to 9-mer, that with an 8-mer DNA adduct most efficiently cleaved RNA site-specifically at 30°C. In this case, the cleavage site was changed, such that the RNA substrate was cleaved at the site, which is located five residues downstream from the 5'-end of the DNA/RNA hybrid. However, it remained to be determined whether a DNA-linked RNase H with altered function can be constructed by using RNases H from different sources, such as RNase HI from Thermus thermophilus HB8, as a catalytic component of DNA-linked RNase H.
T.thermophilus RNase HI is composed of 166 amino acid residues and shows the amino acid sequence identity of 52% to E.coli RNase HI (Kanaya and Itaya, 1992). All the active-site residues identified for E.coli RNase HI (Kanaya, 1998
) are conserved in the T.thermophilus RNase HI sequence. The three-dimensional structure of T.thermophilus RNase HI (Ishikawa et al., 1993
) closely resembles that of E.coli RNase HI (Katayanagi et al., 1990
, 1992
; Yang et al., 1990
), suggesting that the catalytic functions of these two enzymes are similar to each other. Nevertheless, T.thermophilus RNase HI is more stable than E.coli RNase HI by 33.9°C in Tm (Kanaya and Itaya, 1992
). Therefore, a DNA-linked RNase H with T.thermophilus RNase HI as a catalytic component (DNA-linked TRNH) may be useful to cleave RNA in a sequence-specific manner at high temperatures, in which a DNA-linked RNase H with E.coli RNase HI as a catalytic component (DNA-linked ERNH) will be thermally denatured.
In this study, we constructed the d15-C135/TRNH and d15-C135/ERNH, in which the 15-mer DNA with a sequence complementary to the polypurine-tract (PPT) sequence of HIV-1 RNA is attached through a 27 Å linker to Cys135 of the T.thermophilus RNase HI and E.coli RNase HI mutants, respectively. We showed that the d15-C135/TRNH cleaved the 15-mer RNA with the PPT sequence more effectively than the d15-C135/ERNH at temperatures >50°C.
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Materials and methods |
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Competent cells of E.coli HB101 [F, hsdS20(rB, mB), recA13, ara-13, proA2, lacY1, galK2, rpsL20(Smr), xyl-5, mtl-1, supE44, ] was obtained from Takara Shuzo. Plasmid pJAL700T, for the overexpression of T.thermophilus RNase HI, was constructed previously (Kanaya and Itaya, 1992
). E.coli cells were grown in LuriaBertani medium (Miller, 1972
) containing 100 mg/l ampicillin.
Materials
[-32P]ATP (>5000 Ci/mmol) was obtained from Amersham, Crotalus durissus phosphodiesterase from Boehringer Mannheim and acetylated bovine serum albumin from Bethesda Research Laboratories. The 15-mer RNA with a sequence of 5'-AAAAGAAAAGGGGGG-3' (PPT-RNA) was supplied by Takara Shuzo. The 15-mer DNA (5'-CCCCCCTTTTCTTTT-3') complementary to the PPT-RNA (d15-mer) and the d15-mer with aminohexyl (C6) linker at the 5'-terminus (5'-amino-linked DNA) were synthesized by Vector Research. N-(
-Maleimidecaproyloxy)succinimide was obtained from Dojindo Laboratories. PCR primers were synthesized by Sawady Technology. Other chemicals were of reagent grade. The E.coli RNase HI variant C135/ERNH, in which all cysteine residues (Cys13, Cys63 and Cys133) were substituted by Ala and Glu135 was substituted by Cys, was prepared previously (Kanaya et al., 1992
).
Preparation of T.thermophilus RNase HI mutants
Site-directed mutagenesis was carried out by the PCR overlap extension method (Horton et al., 1990) using a 5'-primer with the NdeI site, a 3'-primer with the SalI site and 5' and 3' mutagenic primers. The mutagenic primers were designed so that the codon for Cys13 was changed from TGC to TCG for Ser, the codon for Cys63 was changed from TGC to GCA for Ala and the codon for Arg135 was changed from CGG to TGC for Cys. PCR was performed in 25 cycles with GeneAmp PCR system 2400 (Perkin-Elmer) using KOD polymerase from Toyobo. After the digestion by NdeI and SalI, the PCR products were ligated to the NdeISalI site of plasmid pJAL700T to construct expression vectors for the triple mutant enzyme C135/TRNH with the Cys13
Ser, Cys63
Ala and Arg135
Cys mutations, the double mutant enzyme C13C135/TRNH with the Cys63
Ala and Arg135
Cys mutations and the single mutant enzyme C13/TRNH with the Cys63
Ala mutation alone. The nucleotide sequences of the genes encoding these mutant enzymes were confirmed by the dideoxy chain termination method (Sanger et al., 1977
).
The overproducing strains were constructed by transforming E.coli HB101 with the resultant expression plasmids. Cultivation of the E.coli HB101 transformants with the plasmid pJAL700T derivatives and overproduction and purification of the mutant enzymes were carried out as described previously for those of the wild-type enzyme (Kanaya and Itaya, 1992). The production levels of the mutant enzymes in the E.coli cells were estimated to be 2550 mg/l culture. All of them were purified from the crude lysate of the E.coli cells with a yield of ~60% and 1530 mg of the purified enzymes were obtained from 1 l of culture. The production levels and purities of the mutant enzymes were analyzed by SDSPAGE (Laemmli, 1970
).
Construction of DNA-linked RNases H
The 5'-maleimide-d15-mer was synthesized by the reaction between the 5'-amino-linked DNA and N-(-maleimidecaproyloxy)succinimide as described previously (Kanaya et al., 1992
). Coupling reactions between the C135/TRNH, the C13/TRNH or the C135/ERNH and the 5'-maleimide-d15-mer and the purification of the resultant DNA-linked RNases H (d15-C135/TRNH, d15-C13/TRNH or d15-C135/ERNH, respectively), were also carried out as previously described (Kanaya et al., 1992
). These DNA-linked RNases H have a 27 Å linker [maleimide(CH2)5CONH(CH2)6].
Enzymatic activity
The RNase H activity was determined at 30°C for 15 min in 20 µl of 10 mM TrisHCl (pH 9.0) containing 1 mM MgCl2 and 50 µg/ml acetylated bovine serum albumin (for T.thermophilus enzyme) or 10 mM TrisHCl (pH 8.0) containing 10 mM MgCl2, 50 mM NaCl, 1 mM 2-mercaptoethanol and 50 µg/ml acetylated bovine serum albumin (for E.coli enzyme) by measuring the radioactivity of the acid-soluble digestion product from the substrate, 3H-labeled M13 DNA/RNA hybrid, as described previously (Kanaya et al., 1991). One unit is defined as the amount of enzyme producing 1 µmol/min of acid-soluble material. The specific activity was defined as the enzymatic activity/mg of protein. The protein concentration was determined from the UV absorption at 280 nm, assuming that the mutant enzymes have the same A2800.1% values as those of the wild-type enzymes [1.6 for T.thermophilus RNase HI (Kanaya and Itaya, 1992
) and 2.02 for E.coli RNase HI (Kanaya et al., 1990
)]. The concentrations of the DNA-linked enzymes were also determined from the UV absorption at 280 nm assuming that the molar absorption coefficients of the d15-C135/TRNH, d15-C13/TRNH and d15-C135/ERNH are 1.47x105, 1.47x105 and 1.53x105, respectively. These values are the sum of the molar absorption coefficients of the enzyme (2.9x104 for the T.thermophilus RNase HI derivatives and 3.5x104 for the E.coli RNase HI derivative) and the d15-mer (1.18x105).
Cleavage of oligonucleotide substrate
The PPT-RNA was 32P-labeled at the 5'-end and was used as a substrate representing a single-strand RNA. This substrate (10 pmol) was hydrolyzed with the DNA-linked enzyme or the mixture of the unmodified enzyme and the unlinked d15-mer (molar ratio 1:1) for 15 min at various temperatures in 10 µl of the same buffer as for the digestion of M13 DNA/RNA hybrid. Prior to the reaction, the enzyme solution was incubated at each temperature for 15 min. The hydrolyzates were separated on a 20% polyacrylamide gel containing 7 M urea and were analyzed with Instant Imager (Packard). These hydrolyzates were identified by comparing their migrations on the gel with those of the oligonucleotides generated by the partial digestion of the 32P-labeled PPT-RNA with snake venom phosphodiesterase (Jay et al., 1974). For the determination of the specific activity of the DNA-linked enzyme, the amount of the enzyme was controlled such that the ratio of the substrate hydrolyzed did not exceed 30% of the total. Under this condition, the cleavage of the substrate was linearly dependent on the reaction time, as well as on the enzyme concentration. For the determination of the specific activity (apparent specific activity) of the unmodified enzyme at 30 and 65°C, the substrate was hydrolyzed with 0.3 pmol of the C135/TRNH in the presence of 0.3 pmol of the d15-mer or 0.1 pmol of the C135/ERNH in the presence of 0.1 pmol of the d15-mer. One unit is defined as the amount of enzyme cleaving 1 µmol/min of the substrate. The specific activity was defined as the enzymatic activity/mg of protein.
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Results and discussion |
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DNA-linked ERNH has been constructed by cross-linking a DNA oligomer to the cysteine residue substituted for Glu135 (Kanaya et al., 1992) based on the model for enzyme substrate complex (Nakamura et al., 1991
). The crystal structure of T.thermophilus RNase HI indicates that Arg135 is located at the equivalent position to Glu135 of E.coli RNase HI (Figure 1
). Therefore, we decided to substitute Cys for this arginine residue. In order to cross-link a DNA oligomer specifically to the thiol group at position 135, other free thiol groups should be eliminated. In fact, for the construction of DNA-linked ERNH, all three cysteine residues with free thiol groups were replaced by Ala to make the thiol group of Cys135 unique. T.thermophilus RNase HI contains four cysteine residues at positions 13, 41, 63 and 149. The localizations of these cysteine residues, except for that of Cys149, in the crystal structure of T.thermophilus RNase HI are shown in Figure 1
. However, it seems desirable to leave Cys41 and Cys149 unchanged, because these residues contribute to the protein stability by spontaneously forming a disulfide bond between them in the absence of a reducing reagent (Hirano et al., 1998
). In addition, it has been shown previously for the T.thermophilus RNase HI variant, which lacks the C-terminal 12 residues, that the Cys63
Ala mutation does not seriously affect the enzymatic activity, whereas the Cys13
Ala mutation greatly reduces it (Hirano et al., 1998
). Therefore, we decided to replace Cys13 and Cys63 with Ser and Ala, respectively. The Cys13
Ser mutation is expected to affect the enzymatic activity less seriously than the Cys13
Ala mutation, because the former is more conservative than the latter. In addition to the resultant triple mutant enzyme C135/TRNH, the double and single mutant enzymes C13C135/TRNH and C13/TRNH were constructed to analyze the effect of the individual mutations on the enzymatic activity.
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The activities of the wild-type and mutant enzymes of T.thermophilus RNase HI for the hydrolysis of the M13 DNA/RNA hybrid are summarized in Table I, in comparison with those of E.coli RNase HI. The Cys63
Ala mutation did not seriously affect the enzymatic activity of T.thermophilus RNase HI, as expected. Comparison of the specific activities of the C13/TRNH and C13C135/TRNH indicates that the Arg135
Cys mutation reduced the enzymatic activity of the C13/TRNH by 53%. Likewise, comparison of the specific activities of the C13C135/TRNH and C135/TRNH indicates that the Cys13
Ser mutation reduced the enzymatic activity of the C13C135/TRNH by 84%. Because the Cys13
Ala mutation has been reported to reduce the enzymatic activity of T.thermophilus RNase HI by 83% (Hirano et al., 1998
), the Cys13
Ser and Cys13
Ala mutations reduced the enzymatic activity of T.thermophilus RNase HI by a similar extent. Mutational and structural studies of E.coli RNase HI have suggested that Cys13 is engaged in substrate binding through the formation of a hydrogen bond between the main chain cabonyl oxygen of Cys13 and the substrate (Nakamura et al., 1991
). Slight conformational change of this oxygen atom caused by the mutation may result in a great reduction in the enzymatic activity of T.thermophilus RNase HI. However, these mutations do not seriously affect the catalytic function of E.coli RNase HI (Kanaya et al., 1990
). The reason why the effects of these mutations on the T.thermophilus RNase HI activity are greater than those on the E.coli RNase HI activity remains to be solved.
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Because DNA-linked RNase H cleaves the RNA substrate when its DNA adduct forms a DNA/RNA hybrid with the substrate, any DNA oligomer can be linked to RNase H. In this study, we chose the d15-mer, which is complementary to the PPT sequence of HIV-1 RNA, as a DNA adduct. This PPT sequence is not degraded by the RNase H activity of reverse transcriptase (RT) during the synthesis of the minus-strand DNA and serves as a primer for the synthesis of the plus-strand DNA (Coffin, 1996). We previously constructed the DNA-linked RNase H with a nonadeoxyribonucleotide (d9-mer) adduct (Kanaya et al., 1992
). However, DNA-linked RNases H with the d15-mer adduct is more suitable than those with the d9-mer adduct to compare the activities of DNA-linked ERNH and TRNH, because the 15-bp DNA/RNA hybrid must be more stable than the 9-bp DNA/RNA hybrid. Itakura et al. (1984) have reported that the Tm value of the oligomeric DNADNA duplex increases as its size and GC content increase. The Tm value of the d9-mer/r9-mer hybrid has been reported to be 49°C (Kanaya et al., 1992
), indicating that this substrate is not stable at temperatures >50°C.
The d15-C135/TRNH, d15-C13/TRNH and d15-C135/ERNH were constructed by linking the d15-mer through a 27 Å linker to Cys135 of the C135/TRNH, Cys13 of the C13/TRH and Cys135 of the C135/ERNH, respectively. Comparison of the enzymatic activities of these DNA-linked RNases H for the hydrolysis of the M13 DNA/RNA hybrid with those of the unmodified parent enzymes indicated that cross-linking of the d15-mer to the enzyme greatly reduced the activity (Table I), probably because the DNA adduct interferes with the binding of the DNA/RNA substrate to the enzyme. However, the enzymatic activities of the d15-C135/TRNH and d15-C135/ERNH were ~10% of those of the unmodified parent enzymes, whereas the enzymatic activity of the d15-C13/TRNH was 0.7% of that of the C13/TRNH. The inhibitory effect of the DNA adduct linked to Cys13 is much larger than that linked to Cys135, probably because Cys13 is located more closely to the substrate binding site. A large cleft-like depression, which extends from the negatively charged active-site formed by Asp10, Glu48, Asp70, His124 and Asp134 to the positively charged
III-helix and the following loop (Figure 1
), has been proposed to form a substrate binding site of the enzyme (Nakamura et al., 1991
).
Stability
The thermal stability of the d15-C135/TRNH was compared with that of the d15-C135/ERNH by measuring the residual activities of these enzymes after heating at various temperatures for 15 min, in 10 mM TrisHCl (pH 7.5) containing 0.1 M NaCl, 1 mM EDTA, 10% glycerol and 0.1 mg/ml of bovine serum albumin (Figure 2). The residual activity was determined at 30°C by using the PPT-RNA as a substrate. The d15-C135/TRNH almost fully retained its activity after heating at 90°C. In contrast, the d15-C135/ERNH lost 50% of its activity after heating at 50°C and was almost fully inactivated after heating at 60°C. These results indicate that the d15-C135/TRNH is much more stable than the d15-C135/ERNH.
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To examine whether and how the d15-C135/TRNH, d15-C13/TRNH and d15-C135/ERNH cleave the single-stranded RNA, the 15-base PPT-RNA labeled at its 5'-end was used as a substrate. The cleavage of this substrate with the mixture of the unmodified parent enzyme and the unlinked d15-mer (molar ratio of 1:1) was also examined for comparative purposes. The results obtained at 65°C are shown in Figure 3A and summarized in Figure 3B
, as representatives. The d15-C135/TRNH cleaved the PPT-RNA preferentially at two positions, A6A7 and A8A9, and less preferentially at two other positions, G5A6 and A7A8. Thus, four phosphodiester bonds located between G5 and A9 of the PPT-RNA are susceptible to cleavage with the d15-C135/TRNH. In contrast, an additional four phosphodiester bonds, which are located between A9 and G13 at the 3'-terminal region of the PPT-RNA, were cleaved by the mixture of C135/TRNH and the unlinked d15-mer. These results suggest that the cross-linking of the d15-mer to the enzyme restricts the interaction between the enzyme and the DNA/RNA substrate. The linker between the enzyme and the d15-mer must form a loop in order to bring the DNA/RNA hybrid in contact with the active-site of the enzyme. This loop might prevent the cleavage of the PPT-RNA at its 3'-terminal region, because of steric constraints. In addition, Figure 3B
indicates that the major cleavage site nearest the 5'-end of the PPT-RNA is the phosphodiester bond between the sixth and seventh residues from its 5'-end. This result is consistent with the proposal that the DNA residues complementary to the RNA residues, which are located six or seven residues upstream from the cleavage site, interact with the basic protrusion of the enzyme (Kanaya and Kanaya, 1995
; Iwai et al., 1996
). The cleavage modes of the PPT-RNA with the d15-C135/TRNH and the mixture of the C135/TRNH and d15-mer at different temperatures and those with the d15-C135/ERNH and the mixture of the C135/ERNH and d15-mer at the temperatures <50°C were basically identical with those shown in Figure 3
.
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The d15-C135/TRNH and d15-C135/ERNH showed the highest specific activities for the hydrolysis of the PPT-RNA at 65 and 50°C, respectively (Figure 4). The former was 1.8-fold higher than the latter. As a result, the d15-C135/TRNH cleaves the PPT-RNA more effectively than the d15-C135/ERNH at temperatures >50°C. The optimum temperature of the d15-C135/ERNH is clearly governed by the thermostability of the DNA-linked enzyme. In contrast, the optimum temperature of the d15-C135/TRNH seems to be governed by the stability of the enzyme-linked DNA/RNA hybrid, because the d15-C135/TRNH is highly stable as shown in Figure 2
.
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It is noted that the PPT-RNA/d15-mer hybrid, which must not be cleaved by the RNase H activity of HIV-1 RT, is cleaved by E.coli or T.thermophilus RNase HI. It has been proposed that RNases H recognize a DNA/RNA hybrid as a substrate because it assumes an H-form structure (Fedoroff et al., 1993). The minor groove width in the H-form structure is larger and smaller than those in the B- and A-form structures, respectively (Fedoroff et al., 1993
). However, structural studies have recently shown that a PPT-RNA/DNA hybrid assumes an A-form, instead of an H-form, structure (Xiong and Sundaralingam, 1998
, 2000
). This may be the reason why the RNase H activity of HIV-1 RT cannot cleave a PPT-RNA/DNA hybrid. However, it remained to be determined whether a PPT-RNA/DNA hybrid assumes an H-form structure upon binding to E.coli or T.thermophilus RNase HI or these RNases H recognize the structure of a DNA/RNA hybrid in a less strict manner.
In this study, DNA-linked TRNH has been shown to be more active than DNA-linked ERNH at high temperatures. Therefore, DNA-linked TRNH may be useful for the cleavage of RNA molecules with highly ordered structures. DNA-linked ERNH cannot cleave these RNA molecules, because its DNA adduct cannot hybridize to the target sequence. Thermal denaturation and renaturation of these RNA molecules in the presence of DNA-linked TRNH may facilitate this hybridization and thereby facilitate the sequence-specific cleavage of these RNA molecules, because DNA-linked TRNH will not be denatured at these temperatures.
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Notes |
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Acknowledgments |
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References |
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Received September 1, 2000; revised September 29, 2000; accepted October 12, 2000.