Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination

The bacteriophage P1 Cre recombinase catalyzes site-specific recombination between 34-base-pair loxP sequences in a variety of topological contexts. This reaction is widely used to manipulate DNA molecules in applications ranging from benchtop cloning to genome modifications in transgenic animals. Despite the simple, highly symmetric nature of the Cre-loxP system, there is strong evidence that the reaction is asymmetric; the 'bottom' strands in the recombining loxP sites are preferentially exchanged before the 'top' strands. Here, we address the mechanistic basis for ordered strand exchange in the Cre-loxP recombination pathway. Using suicide substrates containing 5'-bridging phosphorothioate linkages at both cleavage sites, fluorescence resonance energy transfer between synapsed loxP sites and a Cre mutant that can cleave the bridging phosphorothioate linkage but not a normal phosphodiester linkage, we showed that preferential formation of a specific synaptic complex between loxP sites imposes ordered strand exchange during recombination and that synapsis stimulates cleavage of loxP sites.     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression.

2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA. These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H. Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin. Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture.     

Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.

M13 RF IV DNA where phosphorothioate groups are incorporated at restriction endonuclease Nci I recognition sites in the (-)strand is efficiently nicked by the action of this enzyme. Incubation of such nicked DNA with exonuclease III produces gapped DNA. The gap can be filled by reaction with deoxynucleoside triphosphates and DNA polymerase I. When this sequence of reactions is performed with DNA containing a mismatch oligonucleotide primer in the (-)-strand mutational frequencies of 70-90% can be obtained upon transformation. The general nature of this methodology has been further shown to be applicable to other restriction enzymes such as Hind II, Pst I and Fsp I. The mutational frequency obtained using these enzymes is between 40-80% mainly because of less efficient nicking and gapping. Studies on inhibition of Nci I cleavage show that in addition to a phosphorothioate group at the position of cleavage an additional group in the 5'-neighbouring position is necessary for complete inhibition.     

Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays

Model single base extension (SBE) genotyping reactions with individual deoxy-, dideoxy- and acyclonucleoside triphosphates are monitored by MALDI-TOF mass spectrometry. Three non-proofreading DNA polymerases display remarkably high misincorporation (up to 64% of correct incorporation) when extending primers with single substrates at saturating concentrations. Introduction of one phosphorothioate (PS) linkage into the primer 3′ terminus reduces misincorporation by these enzymes an average 1.4-fold (range 0- to 3.5-fold) versus correct incorporation. Combined use of 3′-PS primers with strongly proofreading DNA polymerases yields order of magnitude improvements in SBE fidelity over those produced by the equivalent non-proofreading enzymes. Errors are reduced to below MALDI-TOF detectable levels in almost all cases. The Sp diastereomer of the 3′-PS primer, which can be prepared in situ by incubation with proofreading polymerase, is stable to 3′-exonuclease activity over periods longer than 16 h. Products of correct extension by T7 DNAP are retained over 30–60 min during idling turnover at a dNTP concentration of 2.5 µM, indicating that the assay can be applied over a broad range of substrate concentrations. These results suggest that the use of PS primers and proofreading polymerases will offer a simple and cost-effective means to improve fidelity in a range of single-substrate SBE assay formats.     

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.     

Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha

The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.     

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (2¹⁹) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.     

The role of DNA recombination in herpes simplex virus DNA replication

In many organisms the processes of DNA replication and recombination are closely linked. For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks. Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event. DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombination-dependent replication. In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1). The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed. We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Red(alpha) and beta. The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system. In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed. In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.     

Making AppDNA using T4 DNA ligase

5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions. Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site. This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases. We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.     

Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.     

Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.     

Site-specific recombination intermediates trapped with suicide substrates

A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.     

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.     

Capping DNA with DNA

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

Nucleotide sequence analysis of the linked left and right hand terminal regions of adenovirus type 5 DNA present in the transformed rat cell line 5RK20.

A peculiar phenomenon is observed in several adenovirus type 2 or 5 (Ad2 or Ad5) transformed cell lines: the right hand and left hand terminal regions of the viral genome present in the viral DNA insertions of these cell lines are found to be linked together. A large part of the viral DNA insertion present in the Ad5 transformed rat cell line 5RK20 has been cloned in the lambda vector Charon21A, including the segment containing the linked terminal regions. Sequence analysis of the linkage region showed a perfect homology with the Ad5 DNA sequence and a direct linkage of basepair (bp) 63 of the left hand end of the viral genome to bp 108 of the right hand end. No cellular or rearranged viral sequences were present. Our findings suggest that the joining of viral sequences into the cellular genome.     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.