Studies of the DNA helicase-RNA primase unit from bacteriophage T4. A trinucleotide sequence on the DNA template starts RNA primer synthesis

The purified DNA replication proteins encoded by genes 41 and 61 of bacteriophage T4 catalyze efficient RNA primer synthesis on a single-stranded DNA template. In the presence of additional T4 replication proteins, we demonstrate that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for RNA primer-dependent initiation of new DNA chains. These chains start with primers that have the sequences pppApCpNpNpN and pppGpCpNpNpN, where N can be any one of the four ribonucleotides. Each primer is initiated from the T (A-start primers) or C (G-start primers) in the center of the recognized template sequence. A subset of the DNA chain starts is observed when one of the four ribonucleoside triphosphates used as the substrates for primer synthesis is omitted; the starts observed reveal that both pentaribonucleotide and tetraribonucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive. It was known previously that, when 61 protein is present in catalytic amounts, the 41 and 61 proteins are both required for observing RNA primer synthesis. However, by raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein. The DNA chains made are initiated by primers that seem to be identical to those made when both 41 and 61 proteins are present; however, only those template sites containing the 5'-GCT-3' sequence are utilized. The 61 protein is, therefore, the RNA primase, whereas the 41 protein should be viewed as a DNA helicase that is required (presumably via a 41/61 complex) for efficient primase recognition of both the 5'-GCT-3' and 5'-GTT-3' DNA template sequences.     

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N-terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N-terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys-X2-Cys-X17-Cys-X2-Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4-1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template-directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template-independent fashion. The mutant primase has reduced helicase activity although it catalyzes single-stranded DNA-dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g-atom/mol protein.     

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

46 Effect of guanine substitutions in human telomeric G3(T2AG3)3 DNA sequence

In addition to the well-known Watson–Crick double helix, DNA can form other structures. One of them is a four-stranded quadruplex, formation of which was also acknowledged in in vivo conditions. It was suggested that the presence of quadruplexes in e.g. telomeric region has a significant biological importance. We have studied structural properties of the human telomeric quadruplex formed by G₃(T₂AG₃)₃ and related sequences, in which each guanine base was one-by-one replaced by adenine. In the next step, we have studied sequences, in which two, or even four guanines were replaced by adenine. These sequences were studied in the presence of sodium or potassium ions. Using CD spectroscopy, UV thermal stability measurements, and polyacrylamide gel electrophoresis we found that none of the substitutions hindered the formation of the antiparallel quadruplex formed by the unsubstituted sequence in sodium solutions. However, the effect of substitution differed depending on the position of the guanine replaced. The middle quartet of the antiparallel basket scaffold was the most sensitive and led to the least stable structures. With other sequences, the effect of substitution depends on the position and also on the syn/anti glycosidic bond orientation of the appropriate guanosine in the original quadruplex structure. In the case of the multiple A for G substitutions, the G₃(T₂AG₃)₃ quadruplex was most destabilized by the G:G:A:A tetrad, in which the adenosines substituted syn guanosines. Interestingly, unlike with G₃(T₂AG₃)₃, no structural transitions were observed with the A-containing analogs of the sequence when sodium ions were replaced by potassium ions. The basic quadruplex topology remained antiparallel for all modified sequences in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may be biologically important. In our next studies, we have compared the effect of the G to A substitutions in the human telomere sequence with 8-oxoguanine substituted samples or samples containing guanine apurinic sites. Data obtained from our study show a noticeable trend: it is not the type of the lesion but the position of the modification determines the effect on the conformation and stability of the quadruplex.     

Purification of the 56-kDa component of the bacteriophage T7 primase/helicase and characterization of its nucleoside 5'-triphosphatase activity

Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of a 56- and a 63-kDa species that provides primase and helicase activities for T7 DNA replication. The 56-kDa species has been purified 1800-fold from Escherichia coli cells containing a plasmid that encodes this gene 4 protein. The purified 56-kDa protein is homogeneous, as determined by denaturing gel electrophoresis, and is monomeric in its native form, as indicated by gel filtration. The binding of the 56-kDa protein to single-stranded DNA is stimulated by nucleoside 5'-triphosphates, as is the case for a mixture of the two molecular weight species. In the presence of DNA, the 56-kDa protein preferentially hydrolyzes dTTP (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 396-400). Since nucleoside 5'-triphosphatase activity is necessary for both helicase activity and for translocation of gene 4 protein to primase recognition sites, we have characterized this activity using the 56-kDa protein alone. In the DNA-dependent hydrolysis reaction, the enzyme displays a Km of 10 mM for dTTP, and a Vmax of 2.9 x 10(-5) M/min/mg of protein (at 2.5 micrograms/ml). There is little cooperativity with respect to dTTP binding (Hill coefficient = 1.1) except in the presence of ribonucleoside 5'-triphosphate, an inhibitor of dTTP hydrolysis (Hill coefficient greater than 1.5). The apparent KD for single-stranded circular DNA is 0.2 microM. The active species in dTTP hydrolysis is an oligomer of at least two subunits, as indicated by the effect of enzyme concentration upon the rate of DNA-dependent hydrolysis. The 56-kDa protein also catalyzes DNA-independent hydrolysis of dTTP with a Km of 0.11 mM and a Vmax of 1.3 x 10(-7) M/min/mg of protein (at 8 micrograms/ml). The active species in DNA-independent dTTP hydrolysis is also an oligomer.     

Salt effects on the bacteriophage T7-II structure and activity changes

Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers. The ionic strength range was studied between 20-140 mmol/1. In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength. Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage. Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.     

Synthesis of dicytidylyl-(3'-5')-1,2-di(adenosin-N6-yl)ethane and dicytidylyl-(3'-5')-1,4-di(adenosin-N6-yl)butane: covalently joined terminals of two transfer ribonucleic acids and their behavior toward snake venom phosphodiesterase

The chemical synthesis of the tital bridged trinucleoside diphosphates 3e and 3f along with the corresponding dinucleoside phosphates 3c and 3d is described. Bridged nucleosides 3a and 3b gave on treatment with triethyl orthoformate in the presence of p-toluenesulfonic acid in dimethylformamide the cyclic orthoesters 2a and 2b. Condensation of 2a and 2b with N,2',5'-O-triacetylcytidine 3'-phosphate (1) using dicyclohexylcarbodiimide in pyridine afforded after deblocking and chromatographic separation products 3c-f. The latter were readily degraded with pancreatic RNase, but 3c and 3e were completely resistant toward snake venom phosphodiesterase whereas 3d and 3f were digested to the extent of 65 and 43%, respectively. The major product of degradation of 3f with phosphodiesterase was compound 3d resulting from the combined action of phosphodiesterase and contaminating phosphomonoesterase. The results are explained in terms of stacking of terminal bridge nucleoside units in 3c-f. The implications of these findings for the function of snake venom phosphodiesterase are discussed.     

Synthesis of 5-substituted 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates, their incorporationinto DNA and effects on exonuclease

Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.     

Thermodynamic stability of the 5' dangling-ended DNA hairpins formed from sequences 5'-(XY)2GGATAC(T)4GTATCC-3', where X, Y = A, T, G, C

Expressions for the partition function Q (T) of DNA hairpins are presented. Calculations of Q (T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715-1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two-state manner are required. This method is then applied in a systematic manner to investigate the stability of the six basepair duplex stem 5'-GGATAC-3' having four-base dangling single-strand ends with the sequences (XY)2, where X, Y = A, T, G, C, on the 5' end and a T4 loop on the 3' end. Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0 degrees C above the blunt-ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine greater than T greater than C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two-state melting process. Equally accurate fits of all dangling-ended hairpin melting curves were obtained with the two-state model calculation. This was not the case for the melting curve of the blunt-ended hairpin, indicating the presence of a four-base dangling-end drives hairpin melting to a two-state process. Q (T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q (T), the temperature dependence of the transition enthalpy delta H, entropy delta S, and free energy delta G were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879-3885] on the T2 5' dangling-ended d(GC)3 duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.