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Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.

We have investigated the effect of in vitro transcription on cruciform stability. Replicative form DNA of phiX174 strain ins6240, containing a 48 bp synthetic palindrome in the J-F intercistronic region, was supercoiled in vitro to mean negative superhelical densities (sigma) ranging from 0 to 0.15. The presence of cruciforms was probed by limited digestion with the single-strand specific nuclease Bal31. The 48 bp palindrome was extruded at a mean sigma = -0.05, but only after heating the DNA. An in vitro transcription reaction with E. coli RNA polymerase and [alpha-32P]UTP gave identical transcripts with heated or unheated template DNA. The synthetic cruciform was stable upon binding of the RNA polymerase to the template, but it was destabilized upon movement of the transcription complex along the template. Transcription of unheated templates did not result in cruciform formation. We propose that cruciform structures in supercoiled template DNAs present no hindrance to RNA polymerase, and thus have no detectable effect on transcription elongation in vitro.

The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine. The latter protecting group can be removed under mild conditions with fluoride ion.

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O 6 -methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O 6 -methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecAmediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentrationdependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O 6 -methylguanine residues in methylated DNA by HPLC-MS/MS and 5–10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.

A new restriction endonuclease Abe I has beenisolated from Azotobacter beijerinckii. This enzymerecognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3' and cleaves within it symmetrically at positions -5/-2 in the opposing strands, producing three base protruding 5'-ends.

The need to investigate a complex DNA sample consisting of unknown sequences still prompts the researcher to use in vivo cloning (cloning in bacteria, in phages and in other in vivo systems) as the only way to make the sample suitable for screening and sequencing. Here we demonstrate that it can be easily done by PCR, which saves a lot of effort. The main task of cloning—the amplification of individual molecules—is relatively easy to solve by PCR. It requires the ligation of adapter to all DNA ends in the sample, then dilution of the sample to produce a concentration at which the volume being taken for amplification contains about one molecule, and PCR (‘cloning takes’) with a primer which corresponds to the adapter. However, in this case the amplification products would have the same flanking sequence at both ends and therefore could not be sequenced using these flanks as primer annealing sites—two texts would be read simultaneously. We have developed a technique which, though being close to the aforementioned simple scheme, ensures that the final amplification product is flanked by different adapter sequences at the different ends. Such fragments can be sequenced easily from either of the ends. In our in vitro cloning procedure, we ligate a pair of pseudo double-stranded adapters (1) in a mix to all DNA molecules in the sample. Each adapter is a pair of oligonucleotides of uneven length complementary to each other at the 3′-end of the longer oligo (Fig. 1A), forming a structure that is able to participate in ligation as a normal blunt end. However, only the longer oligo is ligated, because the 5′-ends of both oligos, including the shorter one, lack phosphate groups. The DNA stretch complementary to the ligated longer oligo is produced before the first PCR cycle at temperatures when the smaller oligo already falls off its binding site, but longer DNA molecules still retain their double-stranded structure (in fact, it occurs while the sample is heated prior to the first denaturation stage). The adapters should be rather long (about 40 bp) for two reasons: (i) to make it possible to perform two rounds of PCR with nested primers, which is known to be much more powerful and specific than a one-round amplification; (ii) to suppress the amplification of molecules which had obtained identical adapters at both ends during ligation. Only molecules asymmetrically flanked by the two adapters can be amplified, because after the denaturation of DNA strands the complementary ends of each strand of symmetrically flanked molecules anneal to each other, hiding the primer annealing site before the primer can bind to it. This effect works very effectively even for molecules as long as 5 kb, completely inhibiting the amplification of symmetrically flanked molecules, as was reported recently (1,2). We have already made use of it in several novel PCR–based techniques (1–5). The reliability of the ligation–dilution–amplification scheme as proposed for the amplification of individual molecules has been verified in a model system. The DNA test sample was HaeIII treated φX174 DNA. Figure 1B shows that after the ligation of adapters such a dilution ratio can be selected which produces on average one band per cloning take picked out at random from the original φX174/HaeIII band pattern. We have proved that such a product is asymmetrically flanked by amplifications with oppositely directed primers specific for one of the φX174/HaeIII bands and adapter–specific primers (data submitted but not shown). The asymmetry of the resulting PCR products proves once more that they were amplified from a single molecule: otherwise, the bands almost certainly should have contained fragments of opposing asymmetry (as adapters’ combinations ‘AB’ and ‘BA’ for the same molecule type are equally probable during ligation) and no asymmetry should have been detected in the described experiment. It should be stressed that true ‘single molecule per reaction’ aliquots in the dilution scheme appear statistically only in ∼30% of all takes, even when the dilution ratio is ideally determined. Other aliquots would contain either more than one PCR product or be empty. Therefore, we consider it reasonable to deal with dilutions producing about 10 molecules per cloning take. In this case, statistical fluctuations hinder the analysis much less. After amplification a small number of products in each take can be easily discriminated by electrophoresis. The bands on the gel can be treated as individual clones quite similar to bacterial ones: they can be screened and excised from the gel for separate re–amplification and direct sequencing from adapter–specific primers. Any method designed for sequencing of uncloned PCR products is suitable (for example, 6,7), because the in vitro clones are pure PCR products. In our routine practice we have never encountered any problems with sequencing them. As an example of such an approach, Figure 2 shows pictures of differential screening of a subtracted cDNA sample (prepared as described in 4). The sample was cloned in vitro as described above and a panel of some 50 clones were screened for sequences not present (or present in a much lower concentration) in the reference sample. The band that gave differential signal was picked out and

Bacteriophage phi X174 mutants with insertions of palindromic DNA sequences are rapidly outgrown by competing wild-type phage (Müller & Turnage, J. Mol. Biol. 189: 285). The basis for this defect was investigated and found to be due to an exclusion event early in the infectious cycle, in which phage genomes with palindromic inserts were preferentially excluded by wt phage. In addition, we have obtained further evidence for a palindrome induced genetic instability. Both defects are dependent on palindrome size and sequence, consistent with a model which involves formation of cruciforms, or cruciform-like structures. We propose that formation of unusual DNA secondary structures reduces the effectiveness of replicative form (RF) DNA to interact with limiting replication factors or membrane binding sites, possibly because of interaction with the host recombination system.

Observed frequencies of restriction enzyme cleavage in viral DNAs are compared to those expected from the cleavage site sequence and the known size and nearest neighbour frequencies of the DNAs. It is found that a number of significant deviations from expectation occur, and possible explanations are put forward.

The lysis gene E of bacteriophage PhiX174 has been subjected to deletion and fusion analysis. Deletions of 11 to 90% of gene E specific nucleotides coding for to C-terminal region of the gene product were cloned under transcriptional control of lambda pL. For this purpose plasmid pSU1 was constructed which carries an extended polylinker region downstream of pL. Depending on the number of nucleotides after the last gene E specific codon, various C-terminal segments of protein E were replaced by 4, 5, 53 or 314 unrelated amino acids. Functional analysis for lysis inducing properties of the various gene E mutants revealed that the final 9 codons of the gene could be deleted without loss of function. However, replacements of 19 or more C-terminal codons eliminated gene E activity. Although the functional site of the gene E product is located within the N-terminal half of the polypeptide, the C-terminal part of the protein appears to exhibit severe influence on conformation and/or regulation of the functional site.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.

The phi X174am16 revertant system has been used to investigate the influence of alpha-thio-dNTPs and of Mn2+ on the fidelity of the 9S DNA polymerase alpha from calf thymus. Upon substituting dGTP by alpha-thio-dGTP during the in vitro replication, a nearly tenfold decrease in the frequency of G:G and G:T mispairs is observed. The formation of all other mispairs is not changed in the presence of the corresponding alpha-thio-dNTP. Mn2+ at concentrations of 0.5 mM does not influence the frequencies of the mispairs. The expression rate of errors formed during in vitro replication in the (-) strand has been determined for all mispairs detectable in the phi Xam16 system. The (-) strand expression of G:T, T:T and C:T mismatches is about 50%, whereas for A:G, G:G and C:A mismatches it is clearly below 50%. We conclude that the different base-base mismatches are repaired with different efficiencies.

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.

With biologically active DNA of the bacteriophage phi X174, both single and double-stranded, some physico-chemical and biological parameters of the depurination reaction are studied. It is shown that in single-stranded DNA each apurinic site is lethal, while in double-stranded RFI-DNA only about 5% of these sites are lethal. Furthermore it is concluded that the apurinic sites are formed at different rates in single- and double-stranded DNA and also the conversion into breaks of the apurinic sites is different for both forms of DNA.

A DNA recombining protein has been partly purified from cell lines derived from patients suffering from the hereditary disease, Bloom's syndrome. The protein induces the formation of displacement loops in phi X174 RFI DNA molecules after the addition of single-stranded DNA fragments. A filter binding method and electron microscopy were used to determine the reaction. The recombinogenic protein is dependent on divalent cations and ATP for activity.

The DNA nicking-closing enzyme (type I topoisomerase) from rat liver nuclei breaks single-stranded DNA. The broken strand contains a 5'-hydroxyl and tightly bound protein. The stability of this protein-DNA complex to high salt, alkali and detergent suggests a covalent linkage between the DNA and the enzyme. The observed breakage of single-stranded DNA occurs at neutral pH prior to treatment with alkali or detergent, indicating that the breakage may be the result of an interrupted nicking and closing cycle. The resulting covalent complex could represent a reaction intermediate in the overall nicking-closing reaction.

An apparently homogeneous enzyme preparation, containing an apurinic/apyrimidinic (AP) endonuclease from human placenta, was by DNA sequencing analysis found to act as a class I AP-endonuclease, i.e. produce a 3'-deoxyribose and 5'-phosphomonoester nucleotide termini.

Extracts prepared from phi X174 infected E. coli cells inhibited in vitro RF replication The inhibition was dependent upon the presence of A* protein in the reaction and served as an assay to highly purify the A* protein. Purified A* protein bound tightly to duplex DNA as well as single-stranded DNA. The binding of the A* protein to duplex DNA inhibited (I) its single-stranded DNA specific endonucleolytic activity; (II) in vitro synthesis of viral (+) single stranded DNA on an A-RFII DNA complex template; (III) ATP hydrolysis by rep protein and unwinding of the strands of RF DNA. We propose that this inhibitory activity is responsible in vivo for the shut off of E. coli chromosome replication during phi X174 infection, and has a role in the transition from semiconservative RF DNA replication to single-stranded DNA synthesis in the life cycle of phi X174.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.

Mitomycin C reduced with sodium borohydride induced the DNA damage at deoxyguanosines preferentially in dinucleotide sequence G-T. The DNA damage produced strand breaks when subsequently heated. The DNA damage scarcely occurred when the end-labeled DNA was preincubated with ethidium bromide or actinomycin D before the addition of mitomycin C and the reducing agent. Fully reduced mitomycin C did not induce the DNA damage. The mitomycin C-inducing DNA damage seems to require the intercalation of the partially reduced mitomycin C of short life time, probably semiquinone radical, between DNA base pairs. The inhibitory effects of sodium chloride and radical scavengers suggested that the requirement of the covalent bond formation of mitomycin C to DNA and the involvement of oxygen radicals in the DNA damage. 7-N-(p-hydroxyphenyl)mitomycin C, which is reported to show a higher antitumor activity and a lower toxicity than mitomycin C, was readily reduced with dithiothreitol and induced the sequence-specific DNA damage, whereas mitomycin C was not.

The purified A protein and A* protein of bacteriophage phi X174 have been tested for endonuclease activity on single stranded viral phi X174 DNA. The A protein (55.000 daltons) nicks single-stranded DNA in the same way and at the same place as it does superhelical RFI DNA, at the origin of DNA replication. The A* protein (37.000 daltons) can cleave the single-stranded viral DNA at many different sites. It has however a strong preference for the origin of replication. Both proteins generate 3'OH ends and blocked 5' termini at the nick site.

When using phiX174 RFI DNA as a template, invitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5' end sequences. RNase A digests of alpha(32)P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5' hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5' terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5' end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp).

The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.

The A* protein of bacteriophage phi X174 is a single-stranded DNA specific nuclease. It can cleave phi X viral ss DNA in many different places. The position of these sites have been determined within the known phi X174 nucleotide sequence (1). From the sequences at these sites it is clear that the A* protein recognizes and cleaves at sites that show only partial homology with the origin of RF DNA replication in the phi X DNA. Different parts of the origin sequence can be deduced that function as a signal for recognition and cleavage by the A* protein. We conclude that different parts within the DNA recognition domain of the A* protein are functional in the recognition of the origin sequence in single-stranded DNA. The existence of different DNA recognition domains in the A* protein, and therefore also in the A protein, leads to a model that can explain how the A protein performs its multiple function in the phi X174 DNA replication process (2).

A previously elaborated technique for fixing a chosen partially melted state of DNA with glyoxal was used in a study of the melting process of the replicative form (RF III) of phi X174 DNA. Electron-microscopic maps corresponding to five points of the melting curve of RF III were obtained and compared with the theoretical melting maps obtained in (4) and (6). This comparison clearly shows that only rigorous calculations (4) and not the ones proposed by Azbel (6,7) correctly predict the course of RF III melting.

Purified phi X gene A* protein cleaves phi X single stranded DNA. The cleavage appears to be stoichiometric, whereby a gene A* protein molecule cleaves a phosphodiester bond and binds to the DNA fragment. The size of the cleavage product was inversely proportional to the ratio of A* protein to DNA in the reaction mixture. The cleavage of the DNA resulted in the formation of an A* protein - ssDNA complex identified on SDS-polyacrylamide gels and by banding in CsCl. An A* protein-ssDNA complex was isolated by gel filtration and shown to be active in a ligating reaction in which the two ends of the DNA fragment were joined to form a covalently closed circle. The joining reaction required Mg++ ions and was accompanied by the release of the protein from the DNA.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.

SPLINT (SPLine INTerpolation) is a BASIC microcomputer program that uses electrophoretic data on the migration distances of DNA fragments of known size to approximate the relationship between distance moved and DNA fragment size by a piecewise cubic spline function. This function places all standards exactly on the calibration curve and then determines from it the sizes of other fragments in the gel. Special advantages of this program include the use of a digitizer to enter the fragment positions directly from a gel photograph and the optional generation of a graph of the approximating function plus the placement on it of points from any specified gel lanes.

DNA synthesis catalyzed by E. coli polymerases I or III is inhibited on templates containing N-acetoxy-acetylaminofluorene-reacted adducts. Termination of synthesis occurs just before the site of the adduct. Synthesis on 0X174 templates primed with restriction fragments and treated with AAAF can be visualized on DNA sequencing gels. Comparison of the amounts of the different newly synthesized fragments with those calculated from the probability of termination as determined from the average number of adducts per molecule shows that synthesis terminates, rather than stutters, at each adduct. This method may be useful for detecting the bypass of lesions.

We have used an in vivo plasmid-phi X174 packaging system to detect replication initiation signals in the region of the replication origin (oriC) of the Escherichia coli chromosome. The results obtained are summarized as follows: (i) Neither within nor close to oriC effective signals for initiating complementary strand synthesis could be detected. We conclude that initiation mechanisms for leading and lagging strand synthesis at oriC are not identical to any known priming mechanism of DNA synthesis. (ii) At least five signals that can function as complementary strand origins on ss-plasmid DNA are located in a region about 2000-3300 base pairs away from oriC in the clockwise direction on the chromosome. We suggest that these signals are protein n' like recognition sequences since they are dependent for their activity on dnaB protein and show sequence similarities to other putative n' recognition sequences. Surprisingly, some of the signals are located on the template for leading strand synthesis.

A new procedure for T specific cleavage of DNA fragments utilizing photoreaction with spermine has been described. Irradiation of 3'-[32P]-end-labeled DNA fragments for 10-20 min with a germicidal lamp emitting mainly 254-nm light in the presence of 1 M spermine in distilled water resulted in a T specific cleavage of the DNA chains. This method does not require piperidine treatment. By contrast, when the DNA fragments were irradiated in the presence of methylamine under similar conditions, both G and T bands with the intensity of G greater than T have appeared. A similar but less selective T cleavage has also been observed in the irradiation of 5'-[32P]-end-labeled DNA fragments in the presence of spermine followed by brief heating of the photolysate in a loading buffer for gel electrophoresis. The T specific photoreaction with spermine and the G greater than T reaction with methylamine described here may be conveniently used in combination with the standard Maxam-Gilbert's reactions to provide independent confirmatory readings.

Bacteriophage phi X174 encoded gene A protein is an enzyme required for initiation and termination of successive rounds of rolling circle phi X DNA replication. This enzyme catalyses cleavage and ligation of a phosphodiester bond between nucleotide residues G and A at the phi X origin. The cleavage reaction which occurs during initiation involves formation of a free GOH residue at one end and a covalent bond between tyrosine-OH of the gene A protein and 5' phosphate of the A residue, at the other end of the cleavage site. During termination the covalently bound gene A protein cleaves the phosphodiester bond between G and A at the regenerated origin and ligates the 3' and 5' ends of the displaced genome-length viral DNA to form a circle. Since tyrosyl-OH mediated rearrangements of phosphodiester bonds in DNA may also apply to other enzymes involved in replication or recombination such as topoisomerases we have studied this interesting mechanism in greater detail. Analysis of 32P-labelled gene A protein-DNA complex by tryptic digestion followed by sequencing of 32P-containing peptides showed that two tyrosyl residues in the repeating sequence tyr-val-ala-lys-tyr-val-asn-lys participate in phosphodiester bond cleavage. Either one of these tyrosyl residues can function as the acceptor of the DNA chain. In an alpha-helix the side chains of these tyrosyl residues are in juxtaposition. An enzymatic mechanism is proposed in which these two tyrosyl-OH groups participate in an alternating manner in successive cleavage and ligations which occur during phosphodiester bond rearrangements of DNA.

A type II restriction endonuclease Xmn I with a novel site specificity has been isolated from Xanthomonas manihotis. Xmn I does not cleave SV40 DNA, but cleaves phi X174 DNA into three fragments, which constitute 76.61%, 18.08% and 5.31% of the total length of 5386 base pairs, and cleaves pBR322 DNA into two fragments of 55.71% and 44.29% of the entire 4362 base pairs. The nucleotide sequences around the cleavage sites made by Xmn I are not exactly homologous, but they have a common sequence of 5' GAANNNNTTC 3' according to a simple computer program analysis on nucleotide sequences of phi X174 DNA, pBR322 DNA and SV40 DNA. The results suggest that the cleavage site of Xmn I is located within its recognition sequence of 5' GAANNNNTTC 3'.

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.