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PubMed1994

Unusual conformation of a 3'-thioformacetal linkage in a DNA duplex.

The DNA.DNA duplex d(CGCGTTSCH2OTTGCGC).d(GCGCAAAACGCG) (designated duplex III) containing a 3'-thioformacetal (3'-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH2O) linkage at the central T--T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3'-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the T6(3'-SCH2O)T7 modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3'-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3'-TFMA-modified T6 sugar adopts an O4'-endo form (an intermediate structure between the usual C2'-endo and C3'-endo forms). This change is accompanied by a change in the epsilon (C4'-C3'-S3'-CH2) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6-T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3'-TFMA linkages is merely in the substitution of the T6(O3') in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.

Acetylation↗

PubMed1991

Branch capture reactions: displacers derived from asymmetric PCR.

Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.

Base Sequence↗

PubMed1995

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein.

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

Adenosine Diphosphate↗

PubMed1999

Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases.

We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases [Hang, B., Sági, J., and Singer, B. (1998) J. Biol. Chem. 273, 33406-33413]. This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes. In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability. The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate. When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency. However, the difference in the cleavage rates between different sequences was much less than with the 15-mers. Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site. These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo.

Amino Acid Sequence↗

PubMed2005

Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element.

RNA interference (RNAi) is a powerful method for suppressing the expression of a gene of interest, and can be induced by 21-25 nucleotide small interfering RNA (siRNA) duplexes homologous to the silenced gene, which function as sequence-specific RNAi mediators in RNA-induced silencing complexes (RISCs). In the previous study, it was shown that fork-siRNA duplexes, whose sense-stranded siRNA elements carried a few nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, could enhance RNAi activity more than conventional siRNA duplexes in cultured mammalian cells. In this study, we further characterized fork-siRNA duplexes using reporter plasmids carrying target sequences complementary to the sense- or antisense-stranded siRNA elements in the untranslated region of Renilla luciferase. The data presented here suggest that nucleotide mismatches at either the 3'- or 5'-end of the sense-stranded siRNA elements in fork-siRNA duplexes could influence assembly of not only the antisense-stranded siRNA elements but also the sense-stranded elements into RISCs. In addition, we further suggest the possibility that there could be a positional effect of siRNA duplex on RNAi activity.

3' Flanking Region↗

PubMed1998

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout.

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.

Binding Sites↗

PubMed1993

Targeting in linear DNA duplexes with two complementary probe strands for hybrid stability.

A new in vitro hybridization reaction targets two short complementary RecA protein-coated DNA probes to homologous sequences at any position in a linear duplex DNA molecule. Stable hybrids are obtained after RecA protein removal when both complementary probe strands are present in a four-stranded hybrid, but not when one probe strand is present in a three-stranded hybrid. In four-stranded hybrids with one probe strand biotinylated and the other radiolabelled, the deproteinized hybrids can be isolated and detected by affinity capture on streptavidin-coated magnetic beads. RecA-mediated targeting of complementary biotinylated DNA probe strands allows the affinity capture of 48.5-kilobase duplex lambda genomic DNA. These reactions provide a means of isolating any desired duplex gene or chromosomal DNA fragment.

Adenosine Triphosphate↗

PubMed1986

A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution, and phylogenetic implications.

Restriction endonuclease fragments produced by EcoRI/AvaI or KpnI digestion and containing the small (12S) ribosomal RNA (rRNA) genes from the mitochondrial DNAs (mtDNAs) of the common chimpanzee, pygmy chimpanzee, gorilla, and orangutan were inserted into the plasmids pBR322 or pADD1. After species verification the inserted fragments were digested with SauIIIA, subcloned into M13mp7 vectors, and sequenced. The small rRNA gene sequences were compared with each other and with the published human sequence (Anderson et al. 1981). Substitutions were detected at 118 of the 955 nucleotide positions compared. Pairwise, the sequence differences ranged from 1% (between the chimpanzee species) to 9% (comparisons involving the orangutan); the proportion that were transitions ranged from 87% to 100%. Deletions and/or additions were noted at seven locations. With respect to evolutionary sequence lability, kinetic analysis indicated the presence of at least two classes of nucleotide positions; the more labile class occurs in sequences thought to form self-complementary duplexes (stems) in the mature rRNA. The high frequency of compensating substitutions, which maintain base-pairing within these sequences, corroborates their inferred structure. Phylogenetic inferences drawn from the sequence comparisons support the notion of an approximately equidistant relationship among chimpanzees, gorilla, and man, with the orangutan much less closely related. However, inference from a shared deletion suggests that the gorilla and the chimpanzees may be more closely related to one another than they are to man.

Animals↗

PubMed2003

Binding studies on peptide-oligonucleotide complex: intercalation of tryptophan in GC-rich region of c-myc gene.

Transcriptional regulation of the c-myc gene is essential for normal cellular proliferation; differentiation and overexpression of c-myc is associated with several human cancers. C-myc gene, particularly exon 1, which contains the conserved P1 and P2 promoter regions, has been a potential target for the intercalating drugs in chemotherapy. We have chosen a 21-mer GC-rich oligonucleotide sequence starting from 2281 to 2302 of human c-myc gene located 26 base pair upstream of P1 promoter and partially overlapping with the TATA box of P1. In this paper, we have studied the interaction of a tetrapeptide, KWGK-otBut, with duplex of the above 21-mer sequence under low-salt conditions using UV-Vis absorption, UV melting, fluorescence and circular dichroic (CD) spectroscopy. From the fluorescence quenching data, we determined the two binding constants, K1 (involving only electrostatic interactions) and K2 (involving intercalation), for the formation of (PN)1 and (PN)2 of the two-step mechanism previously established by us. Significant changes were observed in the UV difference absorption spectra and CD spectra of both KWGK and 21-mer duplex upon complex formation even at a very low peptide to nucleotide (P/N) ratios. These spectral changes accompanied by a high value of K2 (=5.13) suggest a strong binding of KWGK involving intercalation of the tryptophan in 21-mer duplex. Based on the above data along with changes observed in deltaH, deltaS and deltaG and increase in melting temperature (by about 8 degrees C) of the 21-mer duplex in presence of KWGK, we propose a model for intercalation of tryptophan of in GC-rich region of c-myc gene. Present observations may be explored in understanding the role of intercalation in protein-nucleic acid interactions in c-myc expression and these results could also help in designing oligopeptides or other low molecular weight ligands to modulate gene expression.

Circular Dichroism↗

PubMed2003

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis.

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.

Animals↗

PubMed1996

Hoogsteen DNA duplexes of 3'-3'- and 5'-5'-linked oligonucleotides and trip formation with RNA and DNA pyrimidine single strands: experimental and molecular modeling studies.

DNA oligonucleotide sequences containing two parallel complementary strands attached through 3'-3' and 5'-5' linkages were synthesized. These oligonucleotides from Hoogsteen base-paired parallel-stranded (PS) hairpin duplexes under appropriate conditions [Kandimalla, E. R., Agrawal, S., Venkataraman, G., & Sasisekharan, V. (1995a) J. Am. Chem. Soc. 117, 6416-6417]. UV melting experiments show that these Hoogsteen hairpin duplexes have a lower thermal stability than that of the Watson-Crick (WC) hairpin duplex (antiparallel) of the same sequence. The circular dichroism (CD) spectrum of the Hoogsteen duplex is different from the canonical B-DNA WC duplex spectrum. The formation of the Hoogsteen duplex is pH-dependent since protonation of cytosine requires lower pH conditions. Studies with oligonucleotides of different loop sizes revel that three- and two-base loops are optimum for the formation of stable Hoogsteen duplexes with 3'-3' and 5'-5' linkages, respectively. The guanine residues in the loop stabilize the duplex as a result of G-G interactions as confirmed by molecular modeling studies. The new PS Hoogsteen duplexes form stable triplexes with complementary (antiparallel to the purine domain) single-stranded RNA and DNA pyrimidine sequences in Py.Pu:Py (pyrimidine third strand-purine WC strand:pyrimidine WC strand) motif. The thermal stability of the resulting triplexes is much higher than that of the conventional triplex (binding of a Hoogsteen pyrimidine third strand to a WC duplex) of the same sequence. The CD spectra of the new triplexes are similar to those of conventional triplexes, suggesting that no conformational change occurs as a result of 3'-3' or 5'-5' linkage. A molecular modeling study was carried out to examine the stereochemical feasibility of the Hoogsteen duplexes and formation of triplexes with single-stranded pyrimidine complementary strands.

Circular Dichroism↗

PubMed1987

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes.

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.

Base Sequence↗

PubMed1993

Orientation isomers of the mitomycin C interstrand cross-link in non-self-complementary DNA. Differential effect of the two isomers on restriction endonuclease cleavage at a nearby site.

Reductively activated mitomycin C (MC) forms DNA interstrand cross-links between two guanines at CG.CG sequences. It is predictable that such cross-links should occur in two isomeric strand orientations in duplex DNA (except when located in the center of a self-complementary duplex). This was verified by the isolation and characterization of a pair of two isomeric oligonucleotides in each case of five non-self-complementary duplexes of 8-bp length, cross-linked by MC. Isomer separation was accomplished by reverse-phase HPLC. The isomers in a pair were formed in approximately 1:1 proportion. Their structures were rigorously characterized by a two-step cross-linking procedure: first, 1''-monoalkylation of each strand, followed by conversion to a cross-linked duplex by annealing the monoalkylated strand to its complement in the presence of a reducing agent. The resulting individual authentic orientation isomers were used as standards for identification of the two isomers formed in the original (one-step) cross-linking reactions. A 16-bp duplex oligonucleotide was synthesized featuring the AluI cognate sequence, separated from a MC cross-link site by only 1 bp. Its two MC cross-linked isomers were prepared separately, and their rate of cleavage by AluI was determined using HPLC. Cleavage of both the unmodified and cross-linked duplexes was nonsymmetrical. The isomer in which the 2''-NH3+ of MC is oriented toward the AluI site was cleaved essentially at the same rate as the control duplex, while cleavage of the isomer with the MC indoloquinone group oriented toward the AluI site was inhibited 2-fold at the faster-cleaved strand.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Prediction of the structure of the Y+.R-.R(+)-type DNA triple helix by molecular modelling.

Molecular mechanics has been used to predict the structure of the Y+.R-.R(+)-type DNA triple helix, in which a second polypurine strand binds antiparallel to the homopurine strand of a homopurine/homopyrimidine stretch of duplex DNA. From calculations on the sequence d(C)10.d(G)10.d(G)10, two likely structures emerge. One has the glycosidic torsions of the third strand bases in the anti-conformation and Hoogsteen hydrogen-bonds to the purine strand of the duplex, the other has the third strand purines in the syn orientation and uses a reverse-Hoogsteen hydrogen-bonding pattern. Despite the large structural differences between these two types of triplex, calculations performed in vacuo with a distance-dependent dielectric constant to mimic the shielding effect of solvent show them to be energetically very similar, with the latter (syn) slightly preferred. However, if explicit solvent molecules are included in the calculation, the anti conformation is found to be much preferred. This difference in the results seems to stem from an underestimation of short-range electrostatic interactions in the in vacuo simulations. When TAA or TAT base triples are substituted for the sixth CGG triple in the sequence, it is found that, for the solvated model, the third strand base of the TAA triple prefers the syn orientation while that in the TAT triple retains a preference, though reduced, for the anti conformation.

Base Sequence↗

PubMed1996

Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications.

Hammerhead ribozymes with long antisense flanks (>50 bases) have been used successfully to inhibit replication of human immunodeficiency virus type 1 (HIV-1) in living cells. To explain their increased efficacy versus antisense controls or catalytically inactive derivatives, one can consider dissociation of the ribozyme-product complex to allow a complete catalytic cycle. In this work we investigated the dissociation of a double-stranded RNA with 56 bp in vitro. Dissociation was observed in the presence of single-stranded RNA with sequence complementarity to one of the duplex strands. A displacement reaction between RNA single strands and the duplex, but not simple dissociation, was strongly suggested by the concentration dependence of this process, the influence of additional non-complementary sequences on the single strand and by the unusually low Arrhenius activation energy. The strand displacement reaction was slow in vitro at 37 degrees C and physiological ionic strength, but was increased to k approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide. This compound is thought to enhance non-sequence-specific association of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that strand displacement can be fast and, more importantly, could be tightly regulated in vivo.

Cetrimonium↗

PubMed2002

AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5'-CACCTGC(N)4/8-3'.

A new type II restriction endonuclease AarI has been isolated from Arthrobacter aurescens SS2-322. AarI recognizes the non-palindromic heptanucleotide sequence 5'-CACCTGC(N)4/8-3' and makes a staggered cut at the fourth and eighth bases downstream of the target duplex producing a four base 5'-protruding end. AarI activity is stimulated by oligodeoxyribonucleotide duplexes containing an enzyme-specific recognition sequence.

Arthrobacter↗

PubMed2005

Srs2 helicase of Saccharomyces cerevisiae selectively unwinds triplet repeat DNA.

Trinucleotide repeat expansions are the mutational cause of at least 15 genetic diseases. In vitro, single-stranded triplet repeat DNA forms highly stable hairpins, depending on repeat sequence, and a strong correlation exists between hairpin-forming ability and the risk of expansion in vivo. Hairpins are viewed, therefore, as likely mutagenic precursors to expansions. If a helicase unwinds the hairpin, it would be less likely to expand. Previous work indicated that yeast Srs2 DNA helicase selectively blocks expansions in vivo (Bhattacharyya, S., and Lahue, R. S. (2004) Mol. Cell. Biol. 24, 7324-7330). For example, srs2 mutants, including an ATPase-defective point mutant, exhibit substantially higher expansion rates than wild type controls. In contrast, mutation of another helicase gene, SGS1, had little effect on expansion rates. These findings prompted the idea that Srs2 might selectively unwind triplet repeat hairpins. In this study, DNA helicase assays were performed with purified Srs2, Sgs1, and Escherichia coli UvrD (DNA helicase II). Srs2 shows substantially faster unwinding than Sgs1 or UvrD on partial duplex substrates containing (CTG) x (CTG) sequences, provided that Srs2 encounters the triplet repeat DNA immediately on entering the duplex. Srs2 was also faster at unwinding (CAG) x (CAG)- and (CCG) x (CCG)-containing substrates and an intramolecular (CTG) x (CTG) hairpin. In contrast, all three enzymes unwind about equally well control substrates with either Watson-Crick base pairs or mismatched substrates with non-CNG repeats. Overall, the selective unwinding activity of Srs2 on triplet repeat hairpin DNA helps explain the genetic evidence that Srs2, not the RecQ homolog Sgs1, is a preferred helicase for preventing expansions.

Adenosine Triphosphatases↗

PubMed1995

Helix-stabilizing agent, CC-1065, enhances suppression of translation by an antisense oligodeoxynucleotide.

The antitumor antibiotic CC-1065 is known to bind at selected sequences in the minor groove of duplex nucleic acids and to hyperstabilize the duplexes against thermal melting. These properties suggested that CC-1065 may enhance translation inhibition by antisense oligonucleotides directed against a specific mRNA. A 585 bp mRNA transcript containing the equine infectious anemia virus (EIAV) S2 gene and a portion of the env gene was prepared. Also, a complementary 20 mer antisense oligodeoxynucleotide (5'-TGTTGGGTAATAGG-GGTTGA-3') was prepared against a target sequence in the mRNA located near the translational initiation sites of the overlapping S2 and env genes. The center of the target sequence had an expected CC-1065 recognition sequence (5'-UAUUA-3'). Translation in the presence of CC-1065 and antisense was markedly suppressed compared with that of antisense alone. Addition of a sense 20 mer strand, with or without CC-1065, had little or no effect on translation. CC-1065 and related compounds may be useful as ligands for enhancing the stability of sense-antisense duplexes and for promoting the inhibition of translation by antisense oligonucleotides.

Antibiotics, Antineoplastic↗