Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation.

As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA. Cleavage results in covalent attachment of VirD2 to the 5' terminal of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation. We demonstrate that this cleavage can be reproduced in vitro: VirD2 protein, the border-cleaving enzyme, was overproduced and purified. Cleavage assays were performed with single-stranded oligodeoxyribonucleotides encompassing the Ti plasmid border region or the transfer origin's nick region of the conjugative plasmid RP4. VirD2 of pTiC58 cleaves both border- and nick region-containing oligonucleotides. However, the relaxase TraI of RP4 can cut only the cognate nick regions. The respective proteins remain covalently bound to the 5' end of the cleavage sites, leaving the 3' termini unmodified. VirD2-mediated oligonucleotide cleavage was demonstrated to be an equilibrium reaction that allows specific joining of cleavage products restoring border and nick regions, respectively. The possible role of VirD2 in T-DNA integration into the plant cell's genome is discussed in terms of less stringent target-sequence requirements.     

In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.

During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.     

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids.

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.     

Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.     

Restriction primer extension method of labeling oligonucleotide probes and its application to the detection of Hb E genes

A new method for labeling oligonucleotides was developed to obtain high specific activity of radioactive probes. In an oligonucleotide molecule, two sequences were designed. One sequence, the 5', contains 19 nucleotides and serves as a template for probe synthesis. The second sequence, 3', contains a consensus sequence which forms a Pst I site after forming a complementary strand with the primer. In the presence of E. coli DNA polymerase I (Klenow fragment), alpha-32P dNTP and other dNTPs, a radioactive labeled oligonucleotide was synthesized by the primer extension method. After Pst I digestion, the probe was different from its template in length by 4 bp and could be separated from each other on urea-polyacrylamide gel electrophoresis (PAGE). A radioactive oligonucleotide probe with extremely high specific activity up to 10(10) dpm/micrograms could be obtained by the use of this method. The oligonucleotide probes have been used for the detection of the Hb E mutation in this report.     

Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.     

Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2.

The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the single-strand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.     

Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I

DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2–695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5′ to the site of cleavage is anchored tightly with extensive noncovalent protein–DNA interactions as predicted by the “enzyme-bridged” model. Distortion of the scissile strand at the -4 position 5′ to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.     

Cleavage specificity of the restriction endonuclease isolated from Haemophilus gallinarum (Hga I).

The nucleotide sequences in the replicative form (duplex) of phiX174 DNA around six sites cut by Hga I, a restriction endonuclease from Haemophilus gallinarum, have been compared. The enzyme produces a staggered cleavage resulting in a pentanucleotide 5'-terminal extension. The sequences within and immediately surrounding the pentanucleotide cleavage site have no obvious relationship. However, the sequence 5'-G-A-C-G-C-3' 3'-C-T-G-C-G-5' occurs five nucleotide pairs to the left of the cut in the upper strand and 10 nucleotide pairs to the left of the cut in the lower strand and, therefore, is believed to constitute the recognition site. This is a member of the class of restriction endonucleases in which recognition and cleavage sites lack 2-fold rotational symmetry. The method used to define the cleavage site is of general applicability.     

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3' end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the "lips" region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.     

Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II).

The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA X Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions.     

Nucleotide sequence at the junction between the coding region of the adenovirus 2 hexon messenger RNA and its leader sequence.

We have determined a 139-base-pair sequence of adenovirus 2 DNA that is located immediately leftwards of the cleavage site for endonuclease Sma I at position 51.1. The established sequence includes the hexon AUG initiator codon, located 75--77 nucleotides leftwards of this cleavage site, and codons for the first 26 amino acids of the hexon polypeptide. By the use of purified hexon mRNA as a template and separated strands of small restriction enzyme fragments as specific primers, the complete 5' noncoding region of the hexon mRNA was synthesized and part of its sequence was determined. The tripartite leader sequence of the hexon mRNA starts 39 nucleotides upstream from the initiator AUG triplet and the total length of the 5' noncoding part of the hexon mRNA was estimated to be 235 nucleotides. The sequence at the junction of the leader sequence permits the formation of secondary structures that may be of importance for the splicing reaction.     

Position-specific trapping of topoisomerase I-DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine

DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1-DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the N(6)-amino group of a central 2'-deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R, 10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3' relative to the scissile strand) from this site. We propose a model with the +1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5' end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1.