Proton relay mechanism of general acid catalysis by DNA topoisomerase IB.

Type IB topoisomerases cleave and rejoin DNA through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr-274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5'-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10(-4.3), was stimulated 2600-fold by a 5'-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5'-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5'-O of the leaving DNA strand.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

Reactions of Cre with Methylphosphonate DNA: Similarities and Contrasts with Flp and Vaccinia Topoisomerase

Background

Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis.

Methodology/Principal Findings

We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step.

Conclusions/Significance

Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.     

Catalytic mechanism of DNA topoisomerase IB

Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 5'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pKa of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.     

Domain structure of vaccinia DNA ligase.

The 552 amino acid vaccinia virus DNA ligase consists of three structural domains defined by partial proteolysis: (i) an amino-terminal 175 amino acid segment that is susceptible to digestion with chymotrypsin and trypsin; (ii) a protease-resistant central domain that contains the active site of nucleotidyl transfer (Lys-231); (iii) a protease-resistant carboxyl domain. The two protease-resistant domains are separated by a protease-sensitive interdomain bridge from positions 296 to 307. Adenylyltransferase and DNA ligation activities are preserved when the N-terminal 200 amino acids are deleted. However, the truncated form of vaccinia ligase has a reduced catalytic rate in strand joining and a lower affinity for DNA than does the full-sized enzyme. The 350 amino acid catalytic core of the vaccinia ligase is similar in size and protease-sensitivity to the full-length bacteriophage T7 DNA ligase.     

Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I.

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.     

Mechanistic plasticity of DNA topoisomerase IB: phosphate electrostatics dictate the need for a catalytic arginine

Four conserved amino acids of type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in vaccinia topoisomerase) catalyze the attack by tyrosine on the scissile phosphodiester to form a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The mechanism entails general acid catalysis (by Lys167 and Arg130) and transition-state stabilization (via contact of His265 with the pro-Sp oxygen). Here we query the function of Arg223, which accelerates transesterification by a factor of 10(5). The requirement for Arg223 is alleviated by a neutral Sp methylphosphonate (MeP) linkage at the cleavage site. Arg223 is not required for the 30,000-fold activation of the latent endonuclease activity of topoisomerase by the Sp MeP. The rate of autohydrolysis by the DNA-(3'-MeP)-topoisomerase intermediate approaches 10% of the rate of religation to a 5'-OH DNA strand. These findings underscore the importance of transition-state electrostatics in determining the composition of the active site and dictating the balance between strand transferase and hydrolase functions.     

The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities.

Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.     

p-Thiophenylalanine-induced DNA cleavage and religation activity of a modified vaccinia topoisomerase IB

Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5'(C/T)CCTTp(↓) sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (k(cl) = 1 × 10(-4) s(-1)) was 30 times lower than for the wild-type enzyme (k(cl) = 3 × 10(-3) s(-1)). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O-P and S-P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg(2+) and Mn(2+) to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg(2+) and Mn(2+) increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion-heteroatom interaction is required for catalysis.     

Identification of a minimal functional linker in human topoisomerase I by domain swapping with Cre recombinase

Cellular forms of type IB topoisomerases distinguish themselves from their viral counterparts and the tyrosine recombinases to which they are closely related by having rather extensive N-terminal and linker domains. The functions and necessity of these domains are not yet fully unraveled. In this study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase. In vitro characterization of the resulting chimeras, denoted Cropos, reveals that six amino acids from the Cre linker loop constitute the minimal length of a functional linker in human topoisomerase I.     

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.     

Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.     

Structural insights on the small subunit of DNA topoisomerase I from the unicellular parasite Leishmania donovani

Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Deletion study of DNA topoisomerase IB from Leishmania donovani: searching for a minimal functional heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved "core" domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIDeltaS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme.     

Remote phosphate contacts trigger assembly of the active site of DNA topoisomerase IB

Vaccinia topoisomerase IB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at its target site 5'-CCCTTp downward arrow in duplex DNA. The contributions of backbone electrostatics and individual phosphate oxygens to the transesterification reaction were probed by introducing 22 single Rp and Sp methylphosphonate diastereomers at 11 positions flanking the cleavage site. Methyl groups at eight positions (four on the scissile strand and four on the nonscissile strand) inhibited the rate of single-turnover cleavage by factors of 50-50,000. Stereospecific interference was observed at several phosphates, thereby distinguishing simple electrostatic contributions from putative specific polar contacts to either the pro-Sp or pro-Rp oxygens. The functionally relevant phosphate oxygens are located on the minor groove face of the helix on which the scissile phosphodiester resides. Our findings, combined with available crystal structures of vaccinia and human topoisomerase IB, show how specific phosphate contacts remote from where chemistry occurs are critical for assembly of the active site.     

Structure-function analysis of Trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism

Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight beta strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 degrees C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 microm) that binds more avidly to the active site than the ATP substrate (Km 25 microm). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.     

Arginine as a General Acid Catalyst in Serine Recombinase-mediated DNA Cleavage

Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3′O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3′ phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3′O leaving group and is the prime candidate for the general acid that protonates the 3′O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.     

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA)

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.     

Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I

Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081; Perry, K., and Mondragón, A. (2003) Structure 11, 1349-1358). Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme. The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage. Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.