Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

A-tract polarity dominate the curvature in flanking sequences

It is well-known, but little understood, that the nucleotide sequences between phased A(4-6)-tracts (at 10-11 bp intervals) have only a slight effect on overall curvature. To explore this phenomenon, we have examined the gel-migration properties of sequences containing both A-tracts as well as G-tracts (i.e., sequences of the form G(n)C(m) or C(n)G(m), n + m > 4) in various relative positioning. We show that the composite bend of these sequences depends on their relative arrangement. When G-tracts are placed between two A-tracts, such that both tracts are repeated in phase to themselves (e.g., G(5)A(6)G(5)A(5)), or adjacent to the 3'-side of A-tracts (e.g., A(6)G(5)N(10)), they have minimal influence on the extent of bending of the composite sequence. When G-tracts are placed one helical repeat away from A-tracts (e.g., G(5)N(5)A(6)N(6)), or are adjacent only to the 5'-side of A-tracts (e.g., G(5)A(6)N(10)) their influence on the composite bend is larger. The differential behavior of AG- versus GA-tracts means that A-tracts influence their flanking sequences in a polar manner. Whereas they suppress, or make constant, the intrinsic bending characteristics of any sequence placed immediately 3' to them (and hence by definition any sequence placed between two phased A-tracts), sequences adjoining them on their 5'-side are free to modulate the overall curvature. We interpret these results as evidence for the dominant nature of the unique and nonuniform structure adopted by tracts of four adenines or more. The effects of A-tracts extend at least five base pairs into the adjoining 3' region. This is further evidence for the complexity of DNA structure and the inadequacy of simple nearest-neighbor models to explain all its manifestations.     

Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats.

The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides. Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units.     

A new model for the bending of DNAs containing the oligo(dA) tracts based on NMR observations.

The conformations of double-stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) and d(GCATTTTGAAACG)d(CGTTTCAAAATGC) have been studied by NMR spectroscopy. Analyses of cross peaks in NOESY spectra between the H2 of an adenine and the H1' of a deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' in each duplex. In view of this gradual compression of the minor groove along the oligo(dA) tract, it can be understood clearly why d(GGAAATTTCC)n x 2 and d(GAAAATTTTC)n x 2 are bent, and d(GGTTTAAACC)n x 2 and d(GTTTTAAAAC)n x 2 are not bent. The relative extents of bending of a series of d(AjN10-j)nd(N10-jTj)n sequences can also be understood systematically. Additionally, it was found that the TA step disturbed the compression of the minor groove of the oligo(dA) tract to some extent.     

Distinct patterns in homooligomer tract sequence context in prokaryotic and eukaryotic DNA

The distributions of the junction sequences of homooligomer tracts of various lengths have been examined in prokaryotic DNA sequences and compared with those of eukaryotes. The general trends in the nearest and next to nearest neighbors to the tracts are similar for both groups. In both prokaryotes and eukaryotes A/T runs are preferentially flanked on either the 5' or the 3' ends by A and/or T. G/C runs are preferentially flanked by G and/or C. There is discrimination against A/T runs flanked by G or C and G/C runs flanked by A or T. However, whereas the distribution of prokaryotic homooligomer tract junction sequences was quite homogeneous, large variations were observed in the 5-fold larger eukaryotic database, increasing in magnitude from tracts of length 2 to 3 to 4 base pairs long. Possible DNA conformational implications and in particular DNA curvature and packaging aspects of prokaryotes and eukaryotes are discussed.     

Structure of d(GT)n repeating sequences with parallel and antiparallel chains]

The ability of oligonucleotides 3'-d(GT)5pO(CH2)5Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[d(GT)]) to form hairpins and higher associates is studied. Optical methods of thermal denaturation and circular dichroism as well as the fluorescence of ethidium bromide and acridine orange bound to oligonucleotides were used. At room temperatures the formation of hairpin structure with parallel and antiparallel strands is possible. Thermodynamic parameters of par[d(GT)] and anti[d(GT)] are similar and equal to delta H = -15 kcal/mol, delta S = -50 cal/mol. deg. In the temperature range 3-10 degrees C par[d(GT)] and anti[d(GT)] form four-stranded structures with parallel chains, in which layers of four G-residues alternate with unpaired T-residues being bulged out easily. On comparison of occurrence of alternating (GT)n, (GC)n and (G)n sequences in genome it can be stated that (GT)n biological functions could be connected with conformational possibilities of the four-stranded parallel structures with unpaired T-residues.     

One- and two-dimensional NMR studies on the conformation of DNA containing the oligo(dA)oligo(dT) tract.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.     

A new model for DNA bending on the basis of gradual compression of the minor groove from 5' to 3' along the oligo(dA) tract

The conformations of double stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) and d(GCATTTTGAAACG).d(CGTTTCAAAATGC) have been studied by means of NMR spectroscopy. Analyses of cross peaks in NOESY spectra between H2 of an adenine and H1' of the deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' along the tract in four oligonucleotides. A new model is proposed as to DNA bending based on the evidence of gradual compression of the minor groove. This model can explain why d(GGAAATTTCC) n x 2 and d(GAAAATTTTC) n x 2 are bent, and d(GGTTTAAACC) n x 2 and d(GTTTTAAAAC) n x 2 are not bent. The bending of d(AjN10-j) n x 2 sequences can also be explained.     

Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA

DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced. The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending. Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent. Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions. We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed. For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite. No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments. The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved. The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang. Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger.     

Anti-mitogenic function of interferon-induced (2'-5')oligo(adenylate) and growth-related variations in enzymes that synthesize and degrade this oligonucleotide

Addition of (2'5')ApApA to concanavalin-A-stimulated mouse spleen lymphocytes strongly inhibits the large increase in RNA and protein synthesis which takes place 24-48 h after stimulation. The inhibitory effect on protein synthesis precedes the effect on RNA synthesis and takes at least 6 h to be detected. Histone synthesis is preferentially inhibited at 48 h. No effect on protein synthesis was detected in unstimulated resting lymphocytes, or in stimulated lymphocytes during the first 24 h after concanavalin A treatment. The anti-mitogenic effect of the (2'-5')oligo(adenylate) seems to result, therefore, from inhibition of protein synthesis taking place before initiation of DNA replication. The mitogenic stimulus produced by the lectin enhances, in lymphocytes, the level of the 2'-phosphodiesterase which degrades (2'-5')oligo(adenylate). Enhancement of the 2'-phosphodiesterase was also observed after serum stimulation of confluent monkey kidney cells. Furthermore, the ratio of (2'-5')oligo(adenylate) synthetase to 2'-phosphodiesterase is ten-times lower in fast-growing kidney cells than in quiescent serum-starved cells. A model for the role of (2'-5')oligo(adenylate) synthesis and degradation in the regulation of cell proliferation by interferon and by mitogens is presented.     

Characterization of divalent cation localization in the minor groove of the A(n)T(n) and T(n)A(n) DNA sequence elements by (1)H NMR spectroscopy and manganese(II)

The localization of Mn(2+) in A-tract DNA has been studied by (1)H NMR spectroscopy using a series of self-complementary dodecamer oligonucleotides that contain the sequence motifs A(n)(n) and T(n)A(n), where n = 2, 3, or 4. Mn(2+) localization in the minor groove is observed for all the sequences that have been studied, with the position and degree of localization being highly sequence-dependent. The site most favored for Mn(2+) localization in the minor groove is near the 5'-most ApA step for both the T(n)A(n) and the A(n)T(n) series. For the T(n)A(n) series, this results in two closely spaced symmetry-related Mn(2+) localization sites near the center of each duplex, while for the A(n)T(n) series, the two symmetry-related sites are separated by as much as one half-helical turn. The degree of Mn(2+) localization in the minor groove of the T(n)A(n) series decreases substantially as the AT sequence element is shortened from T(4)A(4) to T(2)A(2). The A(n)T(n) series also exhibits length-dependent Mn(2+) localization; however, the degree of minor groove occupancy by Mn(2+) is significantly less than that observed for the T(n)A(n) series. For both A(n)T(n) and T(n)A(n) sequences, the 3'-most AH2 resonance is the least broadened of the AH2 resonances. This is consistent with the observation that the minor groove of A-tract DNA narrows in the 5' to 3' direction, apparently becoming too narrow after two base pairs for the entry of a fully hydrated divalent cation. The results that are reported illustrate the delicate interplay that exists between DNA nucleotide sequence, minor groove width, and divalent cation localization. The proposed role of cation localization in helical axis bending by A-tracts is also discussed.     

Montmorillonite,Oligonucleotides, RNA and Origin of Life

Na-montmorillonite prepared from Volclay by the titration method facilitates the self-condensation of ImpA, the 5'-phosphorimidazolide derivative of adenosine. As was shown by AE-HPLC analysis and selective enzymatic hydrolysis of products, oligo(A)s formed in this reaction are 10 monomer units long and contain 67% 3',5'-phosphodiester bonds (Ferris and Ertem, 1992a). Under the same reaction conditions, 5'-phosphorimidazolide derivatives of cytidine, uridine and guanosine also undergo self-condensation producing oligomers containing up to 12-14 monomer units for oligo(C)s to 6 monomer units for oligo(G)s. In oligo(C)s and oligo(U)s, 75-80% of the monomers are linked by 2',5'-phosphodiester bonds. Hexamer and higher oligomers isolated from synthetic oligo(C)s formed by montmorillonite catalysis, which contain both 3',5'- and 2',5'-linkages, serve as catalysts for the non-enzymatic template directed synthesis of oligo(G)s from activated monomer 2-MeImpG, guanosine 5'-phospho-2-methylimidazolide (Ertem and Ferris, 1996). Pentamer and higher oligomers containing exclusively 2',5'-linkages, which were isolated from the synthetic oligo(C)s, also serve as templates and produce oligo(G)s with both 2',5'- and 3',5'-phosphodiester bonds. Kinetic studies on montmorillonite catalyzed elongation rates of oligomers using the computer program SIMFIT demonstrated that the rate constants for the formation of oligo(A)s increased in the order of 2-mer < 3-mer < 4-mer ... < 7-mer (Kawamura and Ferris, 1994). A decameric primer, dA(pdA)8pA bound to montmorillonite was elongated to contain up to 50 monomer units by daily addition of activated monomer ImpA to the reaction mixture (Ferris, Hill and Orgel, 1996). Analysis of dimer fractions formed in the montmorillonite catalyzed reaction of binary and quaternary mixtures of ImpA, ImpC, 2-MeImpG and ImpU suggested that only a limited number of oligomers could have formed on the primitive Earth rather than equal amounts of all possible isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).     

Poly(rC)-binding protein 2 interacts with the oligo(rC) tract of coxsackievirus B3

The human poly(rC)-binding protein (PCBP) 2 is known to interact with enteroviral RNA. Here, the interaction of PCBP2 with RNA target sequences at the 5′ end of the coxsackievirus B3 genome was investigated. Using the electrophoretic mobility shift assay and the yeast three-hybrid system, a short oligo(rC) tract connecting cloverleaf and IRES is demonstrated to contribute to PCBP2 binding. This oligo(rC) tract is conserved among entero- and rhinoviruses. In absence of the viral 3C proteinase, an extended cloverleaf RNA (nt 1-105) containing the oligo(rC) tract interacts with PCBP2 whereas the cloverleaf (nt 1-87) lacking the oligo(rC) tract does not. In the presence of 3C proteinase, cloverleaf RNA (1-87) interacts with PCBP2.     

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.     

Detection and quantification of adducts formed upon interaction of diamminedichloroplatinum (II) with DNA, by anion-exchange chromatography after enzymatic degradation

A method has been developed to determine the adducts formed upon interaction of cis- and trans-diamminedichloroplatinum(II) (cis- and trans-DDP) with DNA. After 5 h at 50 degrees C in the dark, the amount of cis-DDP bound to salmon sperm DNA was larger than the amount of the trans-isomer. After enzymatic degradation with deoxyribonucleases to nucleotides and Pt-containing (oligo)nucleotides, the various products were separated by DEAE chromatography and analyzed for Pt by flameless AAS. Indications were obtained for the presence of nucleotides containing monofunctionally bound Pt and of adducts originating from interstrand DNA crosslinks. DEAE chromatography of digests of cis-DDP-treated DNA yielded a product with overall charge -1, which was identified with NMR and CD as cis-[Pt(NH3)2-d(pGpG)], the oligonucleotide derived from intrastrand crosslinks between two adjacent guanines. Another major peak contained Pt-oligonucleotides with overall charge -2, which could be derived from intrastrand crosslinks between two guanines at sites with pGpXpG (X=T,C,A or G) base sequences.     

Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired.     

Solution structures of the individual single strands of the fragile X DNA triplets (GCC)n.(GGC)n.

Three-dimensional structures of the fragile X triplet repeats (GCC)n and (GGC)n are derived by using one- dimensional/two-dimensional NMR. Under a wide range of solution conditions (10-150 mM NaCl,pH6-7)(GCC)5-7 strands form exclusively slipped hairpins with a 3' overhanging C. The slipped hairpins of (GCC)n strands show the following structural characteristics: (i) maximization of Watson-Crick G.C pairs; (ii) formation of C.C mispairs at the CpG steps in the stem; (iii) C2'-endo, anti conformations for all the nucleotides. The ability of (GCC)n strands to form hairpin structures more readily than complementary (GGC)n strands suggests preferential slippage during replication and subsequent expansion of the (GCC)n strands. In addition, the C.C. mispairs at the CpG site of (GCC)n hairpins account for their exceptional substrate efficiencies for human methyltransferase. Gel electrophoresis data show that (GGC)n strands form both hairpin and mismatched duplex structures in 10-150 mM NaCl (ph 6-7) for n < 10, but for n > or + 11 hairpin structures are exclusively present. However, (GGC)n strands remain predominantly in the duplex state for n=4-11 under NMR solution conditions, which require DNA concentrations 100- to 1000-fold higher than in gel electrophoresis. NMR analyses of [(GGC)n]2 duplexes for n=4-6 show the presence of Watson-Crick G.C and mismatched G anti G syn pairs. The mismatches adjacent to the CpG step introduce local structural flexibility in these duplexes. Similar structural properties are also expected in the stem of the hairpins formed by (GGC)n strands.