Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.     

Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.     

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.     

A rapid and simple method for labeling short DNA fragments using Taq polymerase.

This report describes a new method for labeling PCR-generated short length (60-120 bp) double-stranded DNA fragments for use as hybridization probes. The method utilizes gene-specific primers identical to those for PCR generation of non-radioactive DNA fragments. Radioactive probes are synthesized by Taq DNA polymerase without using PCR. Single-stranded (sense or antisense) and double-stranded probes can be individually prepared by selection of the appropriate primers. The labeling reaction reached maximum incorporation within 30 min with mean specific activities of 1.05 x 10(9) dpm/microgram (antisense single-stranded), and 1.62 x 10(9) dpm/microgram (double-stranded) were obtained using templates 69-117 of nucleotides. This method offers a simple and rapid means of generating antisense probes for Northern blot analyses and double-stranded probes for Southern blot analyses that provide highly intense signals with low background.     

Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.     

The ability of DNA polymerase beta to synthesize DNA beyond the gap with displacement of the non-replicated strand

Study was made of the ability of calf thymus DNA polymerases alpha and beta to replicate templates containing a small gap. It was found that during extensive replication of activated DNA or synthetic template.primers or specially prepared circular DNA containing a small gap, catalyzed by DNA polymerase alpha, the levels of incorporated nucleotides corresponded to the amounts of the single-stranded fraction of these templates. In contrast, in the reaction catalyzed by DNA polymerase beta the amounts of products were several times greater. The ability to synthesize the product in a great excess was a specific feature of the latter enzyme. An analysis of the gap-filling products by sucrose gradient centrifugation, gel electrophoresis and Southern hybridization showed that, contrary to DNA polymerase alpha, DNA polymerase beta exhibited the ability to synthesize DNA not only within but also beyond the gap. The "net" DNA product is complementary to the template strand. It is suggested that DNA was synthesized beyond the gap by displacement of the non-replicated strand.     

Processing of concatemers of bacteriophage T7 DNA in vitro

The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.     

An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure.

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.     

Strand Displacement by DNA Polymerase III Occurs through a ��-��-�� Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of γ-complex to support the reaction in the absence of τ. However, if γ-complex is present to load β₂, a truncated τ protein containing only domains III–V will suffice. This truncated protein is sufficient to bind both the α subunit of DNA polymerase (Pol) III and χψ. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where τ is only required to serve as a scaffold to hold Pol III and χ in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476–23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-τ-ψ-χ-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k_cat/K_m) for the strand displacement reaction is ∼300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (∼300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.     

The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction

A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.     

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.     

Formation of Template-Switching Artifacts by Linear Amplification

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1–2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.     

Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.     

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3′-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.     

Linear nicking endonuclease-mediated strand-displacement DNA amplification

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly.     

Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.     

DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro

We describe a eucaryotic in vitro system for DNA replication derived from Xenopus eggs. In this system, priming and elongation of DNA chains occurs with unusually high efficiency on single-stranded circular DNA templates. Up to 1.5 micrograms M13 DNA can be converted to a completely double-stranded form by 100 microliters egg extract in 1 hr at 22 degrees C, a rate of synthesis comparable with the fastest rates of chromosomal DNA synthesis in early embryogenesis. Initiation of DNA synthesis on double-stranded circular DNA templates was undetectable however. The enzymatic events responsible for complementary-strand synthesis in vitro resemble those presumed to act at the lagging strand of the eucaryotic replication fork in vivo in three ways. First, inhibitor studies indicate that DNA polymerase alpha is required. Second, priming of DNA synthesis by oligoribonucleotides is strongly supported by the complete dependence on ribonucleoside triphosphates in the assay, and the detection of an oligoribonucleotide terminus of 9 or possibly 10 nucleotides associated with nascent DNA chains. Third, the priming reaction is resistant to alpha-amanitin.     

Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.