Nucleic Acids Research, 2000, Vol. 28, No. 9 1893-1898
© 2000 Oxford University Press
DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA
Received January 19, 2000; Revised and Accepted March 15, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5'-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase-based strategies for DNA cloning. Here we characterize a pathway of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3' A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3' G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3' A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3' overhangs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaccinia DNA topoisomerase binds to duplex DNA and incises the phosphodiester backbone of one strand at a specific target site 5'-(C/T)CCTT
(1,2). In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3' phosphate of the incised strand and Tyr-274 of the protein. Topoisomerase can religate the covalently held DNA strand across the same bond originally cleaved (as occurs during relaxation of supercoiled DNA) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule (3,4). Vaccinia topoisomerase can transfer the DNA strand to a 5' OH terminated RNA strand to form a tandem DNA–RNA copolymer (5). Non-nucleic acid nucleophiles can also serve as DNA acceptors (6). The repertoire of DNA-to-DNA joining reactions performed by vaccinia topoisomerase has been studied using synthetic duplex DNA substrates containing a single CCCTT cleavage site. When the substrate is configured such that the scissile bond is situated within 6 bp of the 3' end of the duplex, transesterification is accompanied by spontaneous dissociation of the downstream portion of the cleaved strand. The resulting topoisomerase–DNA complex, containing a 5' single-strand tail on the non-cleaved strand, can religate to an acceptor DNA if the acceptor molecule has a 5' OH tail complementary to that of the activated donor complex (3,4). Sticky-end ligation by vaccinia topoisomerase has been demonstrated using plasmid DNA acceptors with two- or four-base 5' overhangs created by restriction endonuclease digestion (4,7). The topoisomerase discriminates quite well between correctly and incorrectly base-paired 5' overhangs. When the substrate is configured so that the non-scissile strand has a nick opposite the scissile phosphate, topoisomerase cleavage results in dissociation of the entire downstream duplex and the formation of a covalently activated blunt-end donor complex. Topoisomerase bound covalently at a blunt CCCTT duplex end can religate to any 5' OH blunt-end duplex acceptor regardless of its sequence (4).
The specificity of vaccinia topoisomerase in DNA cleavage and its versatility in strand transfer have inspired topoisomerase-based strategies for molecular cloning of DNA (7). Placement of CCCTT sites near both ends of a linear DNA duplex and reaction in vitro with vaccinia topoisomerase results in a bivalent donor complex that can ligate both covalently activated 3' ends to the two free 5' ends of an acceptor DNA with compatible termini to form a circular recombinant (7). The termini for cloning can be composed of complementary 5' overhangs or they can be blunt ends. The covalent donor complex is quite stable (e.g., it remains fully competent for ligation to an exogenous acceptor DNA after incubation for 5 days at 37°C) and can be stored indefinitely at low temperature without loss of activity.
Plasmid vectors activated at both ends by vaccinia topoisomerase are now used widely for DNA cloning (8). Vectors with topoisomerase bound at 3' blunt CCCTT ends can be used to rapidly clone blunt-end restriction fragments or PCR products. Vectors with topoisomerase bound at a CCCTT site in which the 5' end of the non-scissile strand is recessed by 1 nt (leaving a single unpaired 3' T covalently bound to topoisomerase) are used to clone PCR products containing an untemplated 3' nucleotide at both ends. The latter technology (TOPO-TA Cloning, Invitrogen, Carlsbad, CA) has been popularized in the absence of any information in the literature concerning the efficiency, kinetics and specificity of the ligation of 3' overhangs by vaccinia topoisomerase.
Here we use defined donors and acceptors to characterize the 3' overhang pathway of topoisomerase-mediated strand transfer. Our results highlight the contributions of base pairing at the +1T:N position and electrostatic interactions with the acceptor DNA duplex in dictating the rate and extent of recombinant formation in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enzyme purification
Vaccinia topoisomerase was expressed in Escherichia coli BL21 cells infected with bacteriophage
CE6 and then purified from a soluble bacterial lysate by phosphocellulose column chromatography (9). The protein concentration of the phosphocellulose preparation was determined by using the dye-binding method (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard.
DNA substrates and acceptors
A 60mer CCCTT-containing DNA oligonucleotide (Fig. 1) was 5' end-labeled by enzymatic phosphorylation in the presence of [
-32P]ATP and T4 polynucleotide kinase, then purified by preparative electrophoresis through a 20% polyacrylamide gel containing TBE (90 mM Tris-borate, 2.5 mM EDTA). The labeled oligonucleotide was eluted from an excised gel slice and then hybridized to two unlabeled 18mer complementary oligonucleotides at a molar ratio of 60mer/18mer/18mer of 1:4:4 to form the topoisomerase cleavage substrate shown in Figure 1. The 3' N acceptor duplexes with mononucleotide 3' overhangs were prepared by hybridizing equimolar amounts of the complementary 24mer oligonucleotides depicted in Figure 1. The annealing reaction mixtures containing 0.2 M NaCl and the relevant oligonucleotides were heated to 70°C and then slow-cooled to 22°C. The hybridized DNAs were stored at 4°C.
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Topoisomerase-catalyzed strand transfer at a 3' T:N overhang
A reaction mixture containing (per 20 µl) 50 mM Tris–HCl (pH 7.5), 0.5 pmol of 5'-labeled DNA cleavage substrate and 2.5 pmol of topoisomerase was incubated at 37°C for 10 min. An aliquot (20 µl) was then withdrawn and quenched by adding SDS to 0.5% (time 0). Strand transfer was initiated by adding a 50-fold molar excess (25 pmol/20 µl of reaction volume) of acceptor DNA with either 3' A, 3' G, 3' T or 3' C overhangs (Fig. 1) and incubation was continued at 37°C. The acceptors were added either without additional salt or together with salt so as to increase the final salt concentration in the reaction mixtures to 0.5 M NaCl. Aliquots (20 µl) were withdrawn at the times specified and quenched immediately with SDS. The denatured samples were digested with 10 µg of proteinase K for 60 min at 37°C. The samples were adjusted to 33% formamide and heated for 5 min at 95°C. A 10 µl aliquot of each sample was then analyzed by electrophoresis through a 15% polyacrylamide gel containing 7 M urea in TBE. The extent of formation of the 54mer recombinant strand (as a percent of the total radioactivity) was quantitated by scanning the gel with a FUJIX BAS2000 phosphorimager.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Covalently activated donor complex with a 3' T overhang
The DNA substrate used to prepare the donor complex consisted of a 5' 32P-labeled 60mer strand with a centrally placed CCCTT
site annealed to two unlabeled 18mer strands to yield a gapped duplex molecule missing the +1A nucleoside and the flanking +1 and +2 phosphates on the non-scissile strand (Fig. 1). The transesterification reaction of topoisomerase with this substrate resulted in formation of the covalent intermediate depicted in Figure 1 (10). Note that the non-covalently held downstream duplex readily dissociated from the covalent intermediate, leaving the topoisomerase linked to an unpaired 3' T nucleotide. The covalent intermediate was detected after proteinase K digestion of the reaction mixture as a 32P-labeled DNA–peptide adduct migrating at ~32–33 nt during polyacrylamide gel electrophoresis (Fig. 2, lane 0). Seventy five percent of the input 60mer strand was converted to covalent adduct in reactions containing 125 nM topoisomerase and 25 nM substrate.
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Strand transfer to an acceptor duplex with a 3' A overhang
The covalent intermediate with a 3' T overhang was mixed with a 50-fold molar excess of a 23-bp duplex molecule containing a single A nucleotide overhang at both 3' ends (acceptor A in Fig. 1). The 3' A overhang should, in principle, permit base pairing of the acceptor with the 3' T overhang of the donor complex. Transfer of the covalently held 5' 32P-labeled 30mer strand to the unlabeled 24mer strand of the acceptor resulted in the formation of a novel recombinant species consisting of a 54-nt CCCTT strand and a singly nicked complementary strand (Figs 1 and 2). Because the acceptor DNA could be religated in either orientation, the reaction yielded a mixture of two recombinant molecules (Fig. 1 and data not shown).
The recombinant 32P-labeled 54mer accumulated with time to ~60% of the total labeled DNA (Fig. 2). A concomitant decrease in the abundance of the covalent intermediate was consistent with a precursor–product relationship between the covalent complex and the recombinant. Strand transfer was detected as early 10 s after addition of the acceptor and the reaction proceeded to near completion in 10 min. A slow second phase of accumulation of the recombinant from 10 to 60 min probably reflected the formation of additional covalent intermediate on the residual substrate during the incubation and its subsequent reaction with the acceptor DNA. Note the decrease in the abundance of the labeled 60mer during the interval from 10 to 60 min (Fig. 2).
The extent of recombinant formation during a 60 min incubation was measured as a function of the concentration of the 3' A acceptor DNA over a range of 25–1250 nM, corresponding to acceptor/substrate ratios from 1 to 50. The reaction was saturated at an acceptor/substrate ratio of 5 and was half-saturated at equimolar concentrations of acceptor and substrate (not shown).
Strand transfer with mispaired 3' overhangs
To evaluate the importance of base-pairing between the 3' T overhang of the donor complex and the 3' nucleotide of the acceptor, we prepared a series of acceptors composed of an identical 23-bp duplex segment, but differing in the identity of the 3' nucleotide overhang (Fig. 1). Covalent topoisomerase–DNA complexes were reacted separately with acceptors containing a 3' A, G, T or C overhang at both ends. The solution conditions for the strand transfer reactions (50 mM Tris–HCl, no added salt) were unchanged from the conditions during the DNA cleavage reaction. In each case, the covalently held CCCTT strand was transferred to the acceptor to form a 54mer recombinant strand and the extent of strand transfer after 1–3 h was comparable for all four 3' N acceptors (Fig. 3; No NaCl). Thus, the vaccinia topoisomerase can clearly religate 3' overhangs without a stringent requirement for standard base pairing at the +1T position.
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However, there were significant differences in the rates of ligation of paired versus mispaired 3' overhangs by vaccinia topoisomerase (Fig. 4A). The rates of accumulation of recombinant 54mer were obviously slower for the +1T:G and +1T:T mispairs compared to the paired +1T:A configuration, and the +1T:C mispair was slower still. The initial rates of recombinant formation (percent recombinant per second) were calculated from the kinetic data and normalized to a value of 100 for the 3' A acceptor (Table 1). The results showed that mispairing reduced the rate of strand transfer by factors of ~25 for +1T:G, 20 for +1T:T and 100 for +1T:C.
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Stringent reaction conditions that impede 3' mismatch ligation
Non-covalent contacts between vaccinia topoisomerase and the DNA target site are weakened by increasing the ionic strength of the reaction buffer. Addition of 0.5 M NaCl completely blocks the forward DNA cleavage reaction and limits any strand transfer reactions by preformed covalent intermediate to a single round of religation and immediate dissociation from the recombinant product (2,5,11). The effects of high ionic strength on strand transfer to the various 3' N overhang acceptors are shown in Figure 3. In these experiments, the reaction mixtures containing the covalent donor complexes were simultaneously adjusted to 0.5 M NaCl as they were supplemented with a 50-fold molar excess of the 3' N acceptor. The striking finding was that ligation to the mismatched G, T or C acceptors to form a 54mer recombinant was suppressed, whereas ligation to the A acceptor proceeded to virtually the same extent as it did in the reaction lacking added salt (Fig. 3). Thus, high salt conditions make the strand transfer reaction critically dependent on proper +1 T:A base pairing.
We analyzed the kinetics of strand transfer in the presence of 0.5 M NaCl. The paired A acceptor was ligated to the covalently held strand with apparent first order kinetics and the reaction reached an endpoint at which 60% of the input DNA was converted to the 54mer recombinant. A comparison of the initial rates of strand transfer to acceptor A in the absence and presence of salt showed that the rate was slowed by a factor of 34 by 0.5 M NaCl. Because high salt has no effect on transesterification chemistry per se, we infer that this rate decrement reflects the loss of electrostatic contacts between the covalently bound topoisomerase and the duplex acceptor DNA that contribute to the binding and proper positioning of the acceptor DNA nucleophile in the enzyme’s active site.
We detected only trace amounts of strand transfer to the mispaired 3' N acceptors under the stringent reaction conditions (Fig. 4B). The normalized initial rates are shown in Table 1. The magnitudes of the salt inhibition effect on each acceptor were gauged from the ratio of the rates plus and minus salt, as follows: 3' G (240-fold), 3' T (430-fold) and 3' C (130-fold).
The inhibition of 3' N mispair ligation by salt depended on salt concentration. The extent of strand transfer to a 3' G acceptor was unaffected up to 0.1 M NaCl, but decreased progressively at 0.2, 0.3 and 0.5 M NaCl (Fig. 5). In contrast, the extent of ligation to the base-paired A acceptor was relatively impervious to salt.
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Transfer of a covalently activated 3' T overhang to a blunt-end acceptor DNA
The experiments presented above show that base mismatches at the +1T:N position significantly slow the rate of strand transfer by topoisomerase bound at a 3' T overhang. Does this phenomenon reflect the simple absence of a correct T:A base pair or does the mismatch exert an inhibitory effect? To address this question, we assayed strand transfer to a blunt-end 24-bp duplex acceptor DNA under non-stringent reaction conditions. A radiolabeled 54mer recombinant product accumulated with time and the reaction reached an endpoint after 3 h at which ~50% of the input DNA was converted to the recombinant (Fig. 6, No NaCl). Although the yield of product was high, the initial rate of 3' T/blunt ligation was only 1.6% of the initial rate of 3' T/3' A ligation. The kinetic effect of the absence of a nucleoside opposite the covalently bound +1T (an ~60-fold rate decrement) was comparable to the effects of +1T:N mismatches (Table 1). We surmise that the correct +1T:A pair is critical for optimal strand transfer. The rate of 3' T/blunt ligation was reduced by about a factor of 40 by inclusion of 0.5 M NaCl in the religation reaction mixture (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaccinia topoisomerase has proved to be an instructive model system for mechanistic studies of type IB topoisomerases as well as a powerful reagent for molecular cloning of DNA (1,7,8). Here we characterize a mode of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide can rapidly and efficiently transfer the covalently held strand to a duplex DNA acceptor containing a 3' A overhang. The transfer reaction occurs at a lower rate, but still with high efficiency, to DNA acceptors containing mismatched 3' G, T or C overhangs. High selectivity for strand transfer to the correctly paired 3' A acceptor can be imposed by increasing the ionic strength of the religation reaction mixture to 0.5 M NaCl. These findings have mechanistic and practical implications.
The 3' overhang mode of strand transfer by vaccinia topoisomerase differs from the two classes of intermolecular DNA strand transfer reactions described previously (3–5,7) in the following respects. Strand transfer by a covalent donor complex with a 5' single-strand tail on the non-cleaved strand is entirely dependent on base pairing between the exogenous single strand acceptor and the 5' tail of the donor complex in the segment immediately downstream of the scissile phosphate. Ligation to a perfectly paired incoming single strand is both highly efficient and rapid (krel ~1 s–1). The rate of sticky-end ligation is unaffected by high concentrations of salt because the 5' OH strand is hybridized (i.e., it becomes linked in cis) to the tail of the donor complex and this is sufficient to position the 5' OH nucleophile at the active site. In contrast, blunt-end ligation by vaccinia topoisomerase bound at a flush duplex CCCTT end is independent of the sequence of the blunt-end acceptor molecule and the rate of blunt-end ligation is reduced sharply when the salt concentration in the reaction mixture is increased to 
0.5 M NaCl (C.Cheng and S.Shuman, unpublished data). The simplest explanation is that sequence non-specific electrostatic contacts of vaccinia topoisomerase with the phosphodiester backbone downstream of the scissile phosphate are important for productive interaction of an unlinked duplex acceptor DNA with the covalent intermediate and these electrostatic interactions are weakened or disrupted by salt. Similar inferences have been drawn from the differential effects of salt on homology-dependent versus blunt ligation by mammalian topoisomerase I (12,13).
The distinct features of the 3' overhang strand transfer reaction by the donor complex at a 3' T overhang are: (i) its kinetic dependence on base-pairing as well as salt-sensitive contacts; (ii) the fact that base-pairing is required within the CCCTT recognition site; and (iii) the capacity to bias the recombination reaction in favor of the correctly paired ligation partner by simple manipulation of the reaction conditions.
The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation (Table 1) provide a quantitative picture of the relative contributions of +1 T:N base pairing and electrostatic interactions downstream of scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The correct +1 T:A base pair contributes at least a 20-fold enhancement of the rate of strand religation under non-stringent conditions (this being a minimal estimate based on comparing the ligation rates of the 3' A and 3' T acceptors). This value is in accord with kinetic data showing that the +1T:A base pair enhances the forward transesterification reaction (covalent adduct formation) by a factor of 20 compared to the rate of transesterification to a substrate that lacks the +1A on the non-scissile strand (10). Taken together, these results suggest that (i) the +1 base pairing interaction per se helps position the +1 phosphate for nucleophilic attack by Tyr-274 or 5' OH DNA and/or (ii) specific protein contacts with the +1 base pair assist in the formation of an optimal active site.
Salt-sensitive electrostatic interactions of topoisomerase with the 3' A acceptor duplex contribute a 34-fold enhancement of the rate of strand transfer. Under stringent reaction conditions, where electrostatic contacts to the downstream duplex are presumably weakened, the +1 T:A base pair elicits at least a 70-fold enhancement of the rate of strand transfer over the extremely slow rates observed for the blunt-end and mismatched 3' N overhang acceptors (70-fold being a minimal estimate based on comparing the rates for 3' A versus blunt-end acceptors). Nuclease footprinting experiments and modeling of the protein crystal structure on DNA suggest that vaccinia topoisomerase protects an ~10 bp segment downstream of the CCCTT cleavage site (10,14,15). Moreover, the cocrystal structure of human topoisomerase I bound to DNA reveals several protein contacts to the phosphodiester backbone within a 9 bp segment immediately 3' of the scissile phosphate (16,17). Disruption of such contacts likely accounts for the salt effect on 3' overhang ligation.
The ability of vaccinia topoisomerase to transesterify in high yield to a CCCTT site with an unopposed +1T and then rejoin specifically to acceptors with a 3' mononucleotide overhang has engendered an effective method to rapidly clone PCR products containing 3' mononucleotide overhangs, i.e., by positioning the topoisomerase-activated 3' T donor complex on both ends of a linear plasmid vector containing a selectable genetic marker. Upon addition of the PCR-amplified DNA fragment, the activated plasmid vector will spontaneously catalyze ligation of the vector ends to the ends of the insert. Circular recombinants that have incorporated the PCR fragment are then selected by transformation into an appropriate biological host. This method is a variation of the general cloning strategy outlined previously (7), in which the vector ends are activated by topoisomerase transesterification.
The experiments presented above clarify the biochemical underpinnings of the 3' overhang cloning strategy and also reveal a broader specificity in 3' overhang ligation than had been assumed (8), to wit, that TOPO-TA cloning is a subclass of TOPO-TN cloning, where N can be any of the four deoxynucleotides. Although the non-templated 3' nucleotide added during primer extension by DNA polymerases is most frequently 3' A, some DNA polymerases can also add 3' G, C or T. The preference of non-templated 3' A addition versus other 3' additions differs for each polymerase (18). There is not a strict requirement for a 3' A to achieve topoisomerase-mediated cloning of 3' overhangs under non-stringent conditions. This relaxed specificity may enhance the overall efficiency of topoisomerase-mediated PCR fragment cloning. If desired, ligation can be restricted to correctly base-paired 3' overhangs by simply increasing the salt concentration in the reaction mixture.
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ACKNOWLEDGEMENT |
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Supported by NIH Grant GM46330.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 212 639 7145; Fax: +1 212 717 3623; Email s-shuman@ski.mskcc.org
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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