Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.     

Mutational analysis of the 3'-->5' proofreading exonuclease of Escherichia coli DNA polymerase III.

The epsilon subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3'-->5' exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how epsilon performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700-8000-fold enhancement) to moderate (6-20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIepsilon motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of straightepsilon may be specifically involved in binding to the alpha (polymerase) subunit.     

RNA mimetics: oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.     

Synthesis of oligodeoxyribonucleotide N3'-->P5' phosphoramidates.

An efficient synthesis of the novel nucleic acid analogs oligodeoxyribonucleotide N3'-->P5' phosphoramidates, where the 3'-oxygen is substituted by a 3'-nitrogen, is described. Synthesis of the title compounds was accomplished by the following synthetic steps. First, 5'-O-DMT base-protected-3'-amino-2',3'-dideoxynucleosides were prepared. The 3'-aminopyrimidines were obtained via the corresponding 2,3'-anhydronucleosides, whereas 3'-aminopurines were derived via 2'-deoxyxylo precursors. Second, using the prepared 3'-aminonucleosides, oligonucleotide N3'-->P5' phosphoramidates were synthesized on a solid support. Oligonucleotide chain assembly was based upon a carbon tetrachloride-driven oxidative coupling of the appropriately protected 3'-aminonucleosides with the 5'-H-phosphonate diester group, resulting in the formation of an internucleoside phosphoramidate link. Fully deprotected oligonucleotide N3'-->P5' phosphoramidates were characterized by ion exchange and reversed phase HPLC, capillary and slab gel electrophoresis and by 31P NMR analysis. Oligonucleotide N3'-->P5' phosphoramidates form remarkably stable duplexes with complementary RNA strands and also with themselves, where the melting temperature of the complexes exceeded that for the parent phosphodiester compounds by 26-33 degrees C. Additionally, duplexes formed by oligonucleotide phosphoramidates with single-stranded DNA were also more thermally stable than those formed by phosphodiesters. The described properties indicate that these compounds may have great potential in oligonucleotide-based diagnostics and therapeutic applications.     

Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein

Werner syndrome is a human disorder characterized by premature aging, genomic instability, and abnormal telomere metabolism. The Werner syndrome protein (WRN) is the only known member of the RecQ DNA helicase family that contains a 3' --> 5'-exonuclease. However, it is not known whether both activities coordinate in a biological pathway. Here, we describe DNA structures, forked duplexes containing telomeric repeats, that are substrates for the simultaneous action of both WRN activities. We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule. WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end. Progression of the WRN exonuclease is inhibited by the action of WRN helicase converting duplex DNA to single strand DNA on forks of various duplex lengths. The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase. We analyzed the simultaneous action of WRN activities on the long forked duplex in the presence of the WRN protein partners, replication protein A (RPA), and the Ku70/80 heterodimer. RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclease. In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity.     

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.     

alpha-Oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and duplex formation.

The synthesis and hybridization properties of novel nucleic acid analogs, alpha-anomeric oligodeoxyribonucleotide N3'-->P5' phosphoramidates, are described. The alpha-3'-aminonucleoside building blocks used for oligonucleotide synthesis were synthesized from 3'-azido-3'-deoxythymidine or 3'-azido-2',3'-dideoxyuridine via acid catalyzed anomerization or transglycosylation reactions. The base-protected alpha-5'-O-DMT-3'-aminonucleosides were assembled into dimers and oligonucleotides on a solid support using the oxidative phosphorylation method.1H NMR analysis of the alpha-N3'-->P5' phosphoramidate dimer structures indicates significant differences in the sugar puckering of these compounds relative to the beta-N3'-->P5' phosphoramidates and to the alpha-phosphodiester counterparts. Additionally, the ability of the alpha-oligonucleotide N3'-->P5' phosphoramidates to form duplexes was studied using thermal denaturation experiments. Thus the N3'-->P5' phosphoramidate decamer containing only alpha-thymidine residues did not bind to poly(A) and exhibited lower duplex thermal stability with poly(dA) than that for the corresponding beta-anomeric phosphoramidate counterpart. A mixed base decamer alpha-CTTCTTCCTT formed duplexes with the RNA and DNA complementary strands only in a parallel orientation. Melting temperatures of these complexes were significantly lower, by 34-47 or 15-25 degrees C, than for the duplexes formed by the isosequential beta-phosphoramidates in antiparallel and parallel orientations respectively. In contrast, the alpha-decaadenylic N3'-->P5' phosphoramidate formed duplexes with both RNA and DNA complementary strands with a stability similar to that of the corresponding beta-anomeric phosphoramidate. Moreover, the self-complementary oligonucleotide alpha-ATATATATAT did not form an alpha:alpha homoduplex. These results demonstrate the effects of 3'-aminonucleoside anomeric configuration on sugar puckering and consequently on stability of the duplexes.     

Zwitterionic oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and properties.

Zwitterionic, net neutral oligonucleotides containing alternating negatively charged N3'-->P5' phosphoramidate monoester and positively charged phosphoramidate diester groups were synthesized. The ability of zwitterionic phosphoramidates to form complexes with complementary DNA and RNA was evaluated. Stoichiometry and salt dependency of these complexes were determined as a function of the nature of the heterocyclic bases of the zwitterionic compounds. Unlike the melting temperatures of the natural phosphodiester-containing oligomers, the T m of the duplexes formed with the zwitterionic oligothymidylates was salt concentration independent. The thermal stability of these duplexes was much higher with Delta T m values of 20-35 degrees C relatively to phosphodiester counterparts at low salt concentrations. The zwitterionic oligoadenylate formed only 2Py:1Pu triplexes with complementary poly(U) or poly(dT) strands. The thermal stability of these complexes was dependent on salt concentration. Also, the T m values of the complexes formed by the zwitterionic oligoadenylate with poly(U) were 6-41 degrees C higher than for the natural phosphodiester counterpart. Triplexes of this compound with poly(dT) were also more stable with a Delta T m value of 22 degrees C at low salt concentrations. Complexes formed by the zwitterionic oligonucleotides with complementary RNAs were not substrates for RNase H. Surprisingly, the duplex formed by the all anionic alternating N3'-->P5'phosphoramidate-phosphodiester oligothymidylate and poly(A) was a good substrate for RNase H.     

A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins.

Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.     

Oligo-2'-fluoro-2'-deoxynucleotide N3'-->P5' phosphoramidates: synthesis and properties.

Uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 2'-fluoro-2'-deoxy-pyrimidine nucleosides were synthesized using an efficient interphase amidite transfer reaction. The 3'-amino group of solid phase-supported 2'-fluoro-2'-deoxynucleoside was used as an acceptor and 5'-diisopropylamino phosphoramidite as a donor of a phosphoramidite group in the tetrazole-catalyzed exchange reaction. Subsequent oxidation with aqueous iodine resulted in formation of an internucleoside phosphoramidate diester. The prepared oligo-2'-fluoro-nucleotide N3'-->P5' phosphoramidates form extremely stable duplexes with complementary nucleic acids: relative to isosequential phosphodiester oligomers, the melting temperature Tm of their duplexes with DNA or RNA was increased approximately 4 or 5 degrees C per modification respectively. Moreover, these compounds are highly resistant to enzymatic hydrolysis by snake venom phosphodiesterase and they are 4-5 times more stable in acidic media (pH 2.2-5.3) than the parent oligo-2'-deoxynucleotide N3'-->P5' phosphoramidates. The described properties of the oligo-2'-fluoronucleotide N3'-->P5' phosphoramidates suggest that they may have good potential for diagnostic and antisense therapeutic applications.     

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.     

Requirement for the polymerization and 5'-->3' exonuclease activities of DNA polymerase I in initiation of DNA replication at oriK sites in the absence of RecA in Escherichia coli rnhA mutants.

In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.     

A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'--> P5' phosphoramidate linkages.

Non-denaturing gel retardation assay, DNA melting experiments and FTIR spectroscopy were used to characterize the triple helix formed by a 15mer 2'-deoxythymidylate with N3'-->P5'phosphoramidate linkages with its target sequence. The results indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages [dT15(np)] is highly potent to form a triple helix with a dT15*dA15target duplex through Hoogsteenbase-pairing; (ii) it forms a dT15(np)*dA15xdT15(np) triplex with the single-stranded oligo-2'-deoxyadenylate (dA15) without detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15*dA15target duplex, but also partially displaces the dT15 strand from the dT15*dA15duplex to form a dT15(np)*dA15xdT15(np) complex.     

Escherichia coli DbpA is a 3' --> 5' RNA helicase

Previous work has demonstrated that Escherichia coli DbpA is a nonprocessive RNA helicase that can disrupt short RNA helices on either the 5' side or 3' side of hairpin 92 of 23S rRNA. Here the directionality of the helicase activity of DbpA was determined by using substrates containing a short reporter helix in the presence of a second adjacent helix of varying stability placed either 5' or 3' of the reporter helix. When the second helix was on the 5' side of the reporter helix, it had no effect on the dissociation rate of the reporter helix. However, when the second helix was on the 3' side of the reporter helix, its dissociation rate determined the dissociation rate of the reporter helix. This defines DbpA as a 3' --> 5' helicase. Like other helicases, DbpA requires a single-stranded RNA loading site on the 3' side of the duplex for disruption to be observed. Since the loading site could be on either strand of the helix that was disrupted, hairpin 92 does not influence the directionality of the helicase but only aids in targeting RNA substrates.     

Length-dependent degradation of single-stranded 3' ends by the Werner syndrome protein (WRN): implications for spatial orientation and coordinated 3' to 5' movement of its ATPase/helicase and exonuclease domains

Background  

The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN.     

5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein

We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line. The enzyme removes 5' single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed.     

An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification.

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.     

The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.