TERMINAL PHOSPHATE LABELED NUCLEOTIDES: SYNTHESIS,APPLICATIONS, AND LINKER EFFECT ON INCORPORATION BY DNA POLYMERASES

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.     

Nucleotide sequence analysis of DNA. XI. The 3' terminal sequences of bacteriophage lambda and phi 80 DNA

Two procedures have been developed and applied to the determination of the 3′ terminal sequences of λ DNA and φ80 DNA. In the first procedure, each 3′ terminus was specifically labeled with a single ³²P-nucleotide. Radioactive oligonucleotides of different lengths were obtained by partial pancreatic deoxyribonuclease digestion. From the characteristic mobilities of these oligonucleotides in two dimensional fractionation systems, the 3′ terminal sequence -ACCCGCG for the r-strand and -GGTTACG for the l-strand of λ DNA have been determined. In the second procedure, approximately six nucleotides were removed from each 3′ terminus with exonuclease III, and they were replaced with radioactive nucleotides by partial repair synthesis. After enzymatic digestion and sequence analysis, the above sequences have been confirmed. The 3′ terminal sequences in φ80 DNA are identical to those in λ DNA at least up to the fifth nucleotide from the 3′ ends.     

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).     

Recognition site of nuclear factor I, a sequence-specific DNA-binding protein from HeLa cells that stimulates adenovirus DNA replication.

Nuclear factor I is a 47-kd protein, isolated from nuclei of HeLa cells, that binds specifically to the inverted terminal repeat of the adenovirus (Ad) DNA and enhances Ad DNA replication in vitro. We have studied the DNA sequence specificity of nuclear factor I binding using cloned terminal fragments of the Ad2 genome and a set of deletion mutants. Binding of nuclear factor I protects nucleotides 19-42 of Ad2 DNA against DNase I digestion. Filter binding assays show that deletion of the first 23 nucleotides does not impair binding while a deletion of 24 nucleotides reduces binding severely. However, binding studies on Ad12 DNA indicate that nucleotide 24 can be mutated. Fragments containing the first 40 bp are bound normally while the first 38 bp are insufficient to sustain binding. Taken together, these results indicate that the minimal recognition site of nuclear factor I contains 15 or 16 nucleotides, located from nucleotide 25 to nucleotide 39 or 40 of the Ad2 DNA. This site contains two of the four conserved nucleotide sequences in this region. Sequences flanking the minimal recognition site may reduce the binding affinity of nuclear factor I. In accordance with these binding studies, DNA replication of a fragment that carries the sequence of the terminal 40 nucleotides of Ad2 at one molecular end is enhanced by nuclear factor I in an in vitro replication system.     

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Long,Processive Enzymatic DNA Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation.

Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed.     

Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.

Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.     

Progressive structural transitions within Mu transpositional complexes

Assembly of the Mu transpososome is dependent on specific binding sites for the MuA transposase near the ends of the phage genome. MuA also contacts terminal nucleotides but only upon transpososome assembly, and base-specific recognition of the terminal nucleotides is critical for assembly. We show that Mu ends lacking the terminal 5 bp can form transpososomes, while longer DNA substrates with mutated terminal nucleotides cannot. The impact of the mutations can be suppressed by base mismatches near the end of Mu. Deletion of the flanking strands or mutation of the terminal nucleotides has differential effects on the cleavage and strand transfer reactions. These results show that the terminal nucleotides control the assembly and activation of transpososomes by influencing conformational changes around the active site.     

A single mouse alpha-amylase gene specifies two different tissue-specific mRNAs

The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.     

Sequence analysis of heteropolymeric DNA synthesized in vitro by the enzyme terminal deoxynucleotidyl transferase and cloned in Escherichia coli.

We have synthesized in vitro single strands of DNA in reaction mixtures containing the terminal deoxynucleotidyl transferase (Bollum's enzyme), an oligo-dG as primer, the four common deoxynucleoside triphosphates and both Mg and Co ions. The resulting heteropolymers have been converted into double strands, tailed with oligo-dC sequences, annealed with an oligo-dG tailed plasmid vector and cloned in E. coli. Six recombinant plasmids have been isolated and characterized. Two of them have been sequenced. The heteropolymeric chains produced by the terminal transferase, ranging in size between 200 and 400 nucleotides, are richer in purines than in pyrimidines, except in the last portions. Open reading frames for 7-20 amino acids with repeated, in phase translational stop codons are present in these sequences and in their complements.     

Patch homologies and the integration of adenovirus DNA in mammalian cells.

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2-3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5 , corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120-130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca - to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.     

Integration of viral DNA into the genome of the adenovirus type 2-transformed hamster cell line HE5 without loss or alteration of cellular nucleotides

Hamster cell line HE5 has been established from primary LSH hamster embryo cells by transformation with adenovirus type 2 (Ad2) (1). Each cell contains two to three copies of integrated Ad2 DNA (2, 3). We cloned and sequenced the sites of junction between viral and cellular DNAs. The terminal 10 and 8 nucleotides of Ad2 DNA were deleted at the left and right sites of junction, respectively. The integrated viral DNA had an internal deletion between map units 35 and 82 on the Ad2 genome. At the internal site of deletion, the remaining viral sequences were linked via a GT dinucleotide of unknown origin. From HE5 DNA, the unoccupied sequence corresponding to the site of insertion was also cloned and sequenced. Part of this sequence was shown to be expressed as cytoplasmic RNA in HE5 and primary LSH hamster embryo cells. The viral DNA had been inserted into cellular DNA without deletions, rearrangements or duplications of cellular nucleotides at the site of insertion. Thus, insertion of Ad2 DNA appeared to have been effected by a mechanism different from that of bacteriophage lambda in Escherichia coli and from that of retroviral genomes in vertebrates. It was conceivable that the terminal viral protein (4) was somehow involved in integration either on a linear or a circularized viral DNA molecule.     

Activation of DNA primer for DNA polymerase as an ultrasensitive assay for a subgroup of enzymes with deoxyribonuclease activity

Pretreatment of native DNA with nucleases which hydrolyze phosphodiester bonds with liberation of 3′-hydroxyl terminal groups is known to increase the rate of incorporation of nucleotides into DNA by E. coli DNA polymerase I and similar enzymes. Concentration ranges and conditions for this reaction have been established which allow specific detection and quantitative assay of such nucleases. The method permits detection of as little as 0.1 pg of pancreatic deoxyribonuclease, is simple, and requires only commercially available components. It has been utilized to monitor purification of a nuclease from HeLa cell nuclei at concentrations which could not be detected by other methods.