NORMAL GENOMIC

• Chevalier BS, Stoddard BL
• Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
• Nucleic Acids Res. 2001; 29: 3757-74
• Display abstract

• Homing endonucleases confer mobility to their host intervening sequence, either an intron or intein, by catalyzing a highly specific double-strand break in a cognate allele lacking the intervening sequence. These proteins are characterized by their ability to bind long DNA target sites (14-40 bp) and their tolerance of minor sequence changes in these sites. A wealth of biochemical and structural data has been generated for these enzymes over the past few years. Herein we review our current understanding of homing endonucleases, including their diversity and evolution, DNA-binding and catalytic mechanisms, and attempts to engineer them to bind novel DNA substrates.

• Van Roey P, Waddling CA, Fox KM, Belfort M, Derbyshire V
• Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate.
• EMBO J. 2001; 20: 3631-7
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• I-TevI is a site-specific, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove-binding alpha-helix, and a helix-turn-helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix-turn- helix subdomain make hydrophobic contacts, the few base-specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base-specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.

• Bond CS, Kvaratskhelia M, Richard D, White MF, Hunter WN
• Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus.
• Proc Natl Acad Sci U S A. 2001; 98: 5509-14
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• The 2.15-A structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday junction complex is proposed, based on the available functional and structural data.

• Li J et al.
• Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains.
• J Biol Chem. 2000; 275: 41476-86
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• GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

• Wende W et al.
• [Analysis of binding and cleavage of DNA by the gene conversion PI-SCEI endonuclease using site-directed mutagenesis]
• Mol Biol (Mosk). 2000; 34: 1054-64

• Mariconda S, Namgoong SY, Yoon KH, Jiang H, Harshey RM
• Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome.
• J Biosci. 2000; 25: 347-60
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• Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIalpha and IIIbeta, respectively). Although the central domain II (the 'DDE' domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIalpha also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIalpha or IIIbeta domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIalpha or IIIbeta function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.

• Wardleworth BN, Kvaratskhelia M, White MF
• Site-directed mutagenesis of the yeast resolving enzyme Cce1 reveals catalytic residues and relationship with the intron-splicing factor Mrs1.
• J Biol Chem. 2000; 275: 23725-8
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• The Holliday junction-resolving enzyme Cce1 is a magnesium-dependent endonuclease, responsible for the resolution of recombining mitochondrial DNA molecules in Saccharomyces cerevisiae. We have identified a homologue of Cce1 from Candida albicans and used a multiple sequence alignment to predict residues important for junction binding and catalysis. Twelve site-directed mutants have been constructed, expressed, purified, and characterized. Using this approach, we have identified basic residues with putative roles in both DNA recognition and catalysis of strand scission and acidic residues that have a purely catalytic role. We have shown directly by isothermal titration calorimetry that a group of acidic residues vital for catalytic activity in Cce1 act as ligands for the catalytic magnesium ions. Sequence similarities between the Cce1 proteins and the group I intron splicing factor Mrs1 suggest the latter may also possess a binding site for magnesium, with a putative role in stabilization of RNA tertiary structure or catalysis of the splicing reaction.

• Chen JC et al.
• Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding.
• Proc Natl Acad Sci U S A. 2000; 97: 8233-8
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• Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52-288). The structure resolved to 2.8 A is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 A apart. A 26-aa alpha-helix, alpha6, links the C-terminal domain to the catalytic core. A kink in one of the two alpha6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein-DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52-210), independently determined at 1.6-A resolution, is identical to the core domain within the two-domain 52-288 structure.

• Kvaratskhelia M, Wardleworth BN, Norman DG, White MF
• A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc.
• J Biol Chem. 2000; 275: 25540-6
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• Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.

• Reuter M et al.
• Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires.
• J Biol Chem. 1999; 274: 5213-21
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• Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.

• Hagen FK, Hazes B, Raffo R, deSa D, Tabak LA
• Structure-function analysis of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold.
• J Biol Chem. 1999; 274: 6797-803
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• Mucin-type O-glycosylation is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases). Based on sequence relationships with divergent proteins, the ppGaNTases can be subdivided into three putative domains: each putative domain contains a characteristic sequence motif. The 112-amino acid glycosyltransferase 1 (GT1) motif represents the first half of the catalytic unit and contains a short aspartate-any residue-histidine (DXH) or aspartate-any residue-aspartate (DXD)-like sequence. Secondary structure predictions and structural threading suggest that the GT1 motif forms a 5-stranded parallel beta-sheet flanked by 4 alpha-helices, which resembles the first domain of the lactose repressor. Four invariant carboxylates and a histidine residue are predicted to lie at the C-terminal end of three beta-strands and line the active site cleft. Site-directed mutagenesis of murine ppGaNTase-T1 reveals that conservative mutations at these 5 positions result in products with no detectable enzyme activity (D156Q, D209N, and H211D) or <1% activity (E127Q and E213Q). The second half of the catalytic unit contains a DXXXXXWGGENXE motif (positions 310-322) which is also found in beta1,4-galactosyltransferases (termed the Gal/GalNAc-T motif). Mutants of carboxylates within this motif express either no detectable activity, 1% or 2% activity (E319Q, E322Q, and D310N, respectively). Mutagenesis of highly conserved (but not invariant) carboxylates produces only modest alterations in enzyme activity. Mutations in the C-terminal 128-amino acid ricin-like lectin motif do not alter the enzyme's catalytic properties.

• Silva GH, Dalgaard JZ, Belfort M, Van Roey P
• Crystal structure of the thermostable archaeal intron-encoded endonuclease I-DmoI.
• J Mol Biol. 1999; 286: 1123-36
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• I-DmoI is a 22 kDa endonuclease encoded by an intron in the 23 S rRNA gene of the hyperthermophilic archaeon Desulfurococcus mobilis. The structure of I-DmoI has been determined to 2.2 A resolution using multi-wavelength anomalous diffraction techniques. I-DmoI, a protein of the LAGLIDADG motif family, represents the first structure of a freestanding endonuclease with two LAGLIDADG motifs, and the first of a thermostable homing endonuclease. I-DmoI consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) related by pseudo 2-fold symmetry. The LAGLIDADG motifs are located at the carboxy-terminal end of the first alpha-helix of each domain. These helices form a two-helix bundle at the interface between the domains and are perpendicular to a saddle-shaped DNA binding surface, formed by two four-stranded antiparallel beta-sheets. Despite substantially different sequences, the overall fold of I-DmoI is similar to that of two other LAGLIDADG proteins for which the structures are known, I-CreI and the endonuclease domain of PI-SceI. The three structures differ most in the loops connecting the beta-strands, relating to the respective DNA target site sizes and geometries. In addition, the absence of conserved residues surrounding the active site, other than those within the LAGLIDADG motif, is of mechanistic importance. Finally, the carboxy-terminal domain of I-DmoI is smaller and has a more irregular fold than the amino-terminal domain, which is more similar to I-CreI, a symmetric homodimeric endonuclease. This is reversed compared to PI-SceI, where the amino-terminal domain is more similar to carboxy-terminal domain of I-DmoI and to I-CreI, with interesting evolutionary implications.

• Tsutakawa SE et al.
• Crystallographic and functional studies of very short patch repair endonuclease.
• Mol Cell. 1999; 3: 621-8
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• Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.

• Shliapnikov SV et al.
• [Serratia marcescens extracellular endonuclease. II. Amino acid residues of the active site and hypothetical mechanism of enzyme function]
• Mol Biol (Mosk). 1999; 33: 454-61

• Gual A, Alonso JC
• Characterization of the small subunit of the terminase enzyme of the Bacillus subtilis bacteriophage SPP1.
• Virology. 1998; 242: 279-87
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• The small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix-turn-helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene 1 of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure.

• Gimble FS, Duan X, Hu D, Quiocho FA
• Identification of Lys-403 in the PI-SceI homing endonuclease as part of a symmetric catalytic center.
• J Biol Chem. 1998; 273: 30524-9
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• Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed to bind DNA. At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously. Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme. Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity. Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA.

• Birkenbihl RP, Kemper B
• Localization and characterization of the dimerization domain of holliday structure resolving endonuclease VII of phage T4.
• J Mol Biol. 1998; 280: 73-83
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• Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4. Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA. In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated. Some of these mutations affected the dimerization of Endo VII. Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state. Only the dimeric fraction of this protein bound to DNA. The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde. The dimerization domain was thus determined to reside between amino acid residues 55 and 105. Computer analyses predicted two alpha-helices (H2 and H3) in this section of the protein. As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization. Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them.

• Flick KE, Jurica MS, Monnat RJ Jr, Stoddard BL
• DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI.
• Nature. 1998; 394: 96-101
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• Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.

• Hwang KY, Baek K, Kim HY, Cho Y
• The crystal structure of flap endonuclease-1 from Methanococcus jannaschii.
• Nat Struct Biol. 1998; 5: 707-13
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• Flap endonuclease-1 (FEN-1), a structure specific nuclease, is an essential enzyme for eukaryotic DNA replication and repair. The crystal structure of FEN-1 from Methanococcus jannaschii, determined at 2.0 A resolution, reveals an active site with two metal ions residing on top of a deep cleft where several conserved acidic residues are clustered. Near the active site, a long flexible loop comprised of many basic and aromatic residues forms a hole large enough to accommodate the DNA substrate. Deletion mutations in this loop significantly decreased the nuclease activity and specificity of FEN-1, suggesting that the loop is critical for recognition and cleavage of the junction between single and double-stranded regions of flap DNA.

• Pingoud V, Grindl W, Wende W, Thole H, Pingoud A
• Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.
• Biochemistry. 1998; 37: 8233-43
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• PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

• Lykke-Andersen J, Garrett RA, Kjems J
• Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.
• EMBO J. 1997; 16: 3272-81
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• Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.

• Clubb RT, Schumacher S, Mizuuchi K, Gronenborn AM, Clore GM
• Solution structure of the I gamma subdomain of the Mu end DNA-binding domain of phage Mu transposase.
• J Mol Biol. 1997; 273: 19-25
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• The MuA transposase of phase Mu is a large modular protein that plays a central role in transposition. We show that the Mu end DNA-binding domain, I beta gamma, which is responsible for binding the DNA attachment sites at each end of the Mu genome, comprises two subdomains, I beta and I gamma, that are structurally autonomous and do not interact with each other in the absence of DNA. The solution structure of the I gamma subdomain has been determined by multidimensional NMR spectroscopy. The structure of I gamma comprises a four helix bundle and, despite the absence of any significant sequence identity, the topology of the first three helices is very similar to that of the homeodomain family of helix-turn-helix DNA-binding proteins. The helix-turn-helix motif of I gamma, however, differs from that of the homeodomains in so far as the loop is longer and the second helix is shorter, reminiscent of that in the POU-specific domain.

• Datta AK, Paulson JC
• Sialylmotifs of sialyltransferases.
• Indian J Biochem Biophys. 1997; 34: 157-65
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• The sialyl moiety of sialylated glycoconjugates expressed on the cell surface are increasingly recognized as the key determinants of various biological recognition events. The transfer of sialic acid to these glycoconjugates are catalyzed by sialyltransferases, a group of 15 or more Golgi enzymes. Cloning of three sialyltransferases from this laboratory, indicated for the first time, that these enzymes are type II membrane proteins and share the topological features common to other glycosyltransferases. However, unlike the other members of the glycosyltransferase family, these enzymes showed the presence of two conserve protein domains, termed 'sialylmotifs'. This unique feature was subsequently found to be present in all the sialyltransferases cloned to-date. The larger 'L-sialylmotif' consisting of 48-49 amino acids is present in the middle of the luminal catalytic domain and has, eight invariant residues, while the 'S-sialylmotif' present closer to the C-terminal end of the enzyme has two invariants among a stretch of 23 amino acids. The other not-so-invariant amino acids are also conserved and their replacement is limited. The functional role of these two sialylmotifs were investigated by single-point site-directed mutagenesis using Gal beta 1, 4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) as a model. Detailed kinetic analysis of the mutants indicated that the 'L-sialylmotif' contributes to the binding of the common donor substrate CMP-NeuAc, while the 'S-sialylmotif' contributes to the binding of both the donor and acceptor substrates.

• Giraud-Panis MJ, Lilley DM
• T4 endonuclease VII. Importance of a histidine-aspartate cluster within the zinc-binding domain.
• J Biol Chem. 1996; 271: 33148-55
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• The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc-binding region toward the N-terminal end of the primary sequence. In the center of this 39-amino acid section (between residues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster. Closely related sequences are found in three other proteins that have similar zinc-binding motifs. We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single amino acids and studying the properties of the resulting N-terminal protein A fusions. No sequence changes within the His-acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc. We found that the N-terminal aspartate residue (Asp-40) and the two histidine residues (His-41 and His-43) within the cluster are essential for junction-cleavage activity of the proteins. However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enzymes. We conclude that the process of cleavage can be uncoupled from those of binding to and distortion of the junction. It is probable that some amino acid side chains of the His-acid cluster participate in the phosphodiester cleavage mechanism of endonuclease VII. The essential aspartate residue might be required for coordination of catalytic metal ions.

• Friedhoff P, Kolmes B, Gimadutdinow O, Wende W, Krause KL, Pingoud A
• Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.
• Nucleic Acids Res. 1996; 24: 2632-9
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• Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.

• Bryk M, Belisle M, Mueller JE, Belfort M
• Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease.
• J Mol Biol. 1995; 247: 197-210
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• I-TevI, a double-strand DNA endonuclease involved in the mobility of the td intron of phage T4, is highly unusual in that it binds and cleaves intronless td alleles (td homing sites) in a site-specific but sequence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 and 25 nucleotides away on the top and bottom strands, respectively. Mapping studies indicate that I-TevI has both sequence and distance sensors that function during cut-site selection. Although I-TevI cleavage of many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively span two turns of the helix, indicating that the interaction is extraordinarily flexible. However, the endonuclease does exhibit spacing preferences between its binding domains, and sequence preferences near the cleavage site, with the G:C pair at -23 implicated as a cleavage determinant. Furthermore, I-TevI appears to function through interactions across the minor groove at the cleavage site, as it does at the intron insertion site, and to be capable of cleaving sequentially, first on the bottom and then on the top strand. These properties of I-TevI are incorporated in a model wherein the endonuclease effects distant cleavage via a flexible hinge.

• Mueller JE, Smith D, Bryk M, Belfort M
• Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.
• EMBO J. 1995; 14: 5724-35
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• I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion. We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change. These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages.

• Bryk M, Quirk SM, Mueller JE, Loizos N, Lawrence C, Belfort M
• The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.
• EMBO J. 1993; 12: 2141-9
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• I-TevI, a double-strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I-TevI, all three domains are important for formation of a cleavage-competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I-TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I-TevI recognizes T-even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.

• Dodson ML, Schrock RD 3rd, Lloyd RS
• Evidence for an imino intermediate in the T4 endonuclease V reaction.
• Biochemistry. 1993; 32: 8284-90
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• Reductive methylation and site-directed mutagenesis experiments have implicated the N-terminal alpha-amino group of T4 endonuclease V in the glycosylase and abasic lyase activities of the enzyme. NMR studies have confirmed the involvement of the N-terminal alpha-amino group in the inhibition of enzyme activity by reductive methylation. A mechanism accounting for these results predicts that a (imino) covalent enzyme-substrate intermediate is formed between the protein N-terminal alpha-amino group and C1' of the 5'-deoxyribose of the pyrimidine dimer substrate subsequent to (or concomitantly with) the glycosylase step. Experiments to verify the existence of this intermediate indicated that enzyme inhibition by cyanide was substrate-dependent, a result classically interpreted to imply an imino reaction intermediate. In addition, sodium borohydride reduction of the intermediate formed a stable dead-end enzyme-substrate product. This product was formed whether ultraviolet light-irradiated high molecular weight DNA or duplex oligonucleotides containing a defined thymine-thymine cyclobutane dimer were used as substrate. The duplex oligonucleotide substrates demonstrated a well-defined gel shift. This will facilitate high-resolution footprinting of the enzyme on the DNA substrate.

• Bell-Pedersen D, Quirk SM, Bryk M, Belfort M
• I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.
• Proc Natl Acad Sci U S A. 1991; 88: 7719-23
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• Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.