Cleavage of Bacteriophage f1 DNA by the Restriction Enzyme of Escherichia coli B

We studied the cleavage of the replicative-form DNA (RF I) of bacteriophage f1 and its SB mutants by purified restriction endonuclease of E. coli B. The results indicate that: (i) Circular replicative forms are broken once to yield full-length linear molecules (RF III). Such linear molecules are less susceptible than RF I to endonuclease R-B. (ii) The genetic sites (SB sites) that confer on the DNA susceptibility to B-restriction are not the actual sites of cleavage. The number of possible cleavage sites is larger than the number of SB sites. We conclude this because an RF III molecule produced by endonuclease R-B from RF I of a mutant that has only one SB site can be circularized by denaturation and renaturation. (iii) The SB site is not modified when the DNA is cleaved, since an SB site can be used repeatedly by endonuclease R-B; the RF III described in ii can be cleaved by the same enzyme after denaturation and renaturation.     

Cis-Limited Action of the Gene-A Product of Bacteriophage ϕX174 and the Essential Bacterial Site

Parental replicative-form (RF(*)) DNA of bacteriophage varphiX174 in a replication-deficient host cell (rep(3) (-)) exhibits two characteristic features that correlate the function of viral gene A with the initiation of viral DNA replication: a specific discontinuity in the viral strand of a constant number of RF molecules and elongation of the viral strand to yield replicative-intermediate DNA forms with single-stranded tails. At high multiplicities of infection, these initiation events are limited to an average of four specifically nicked RFII molecules per cell. The limiting factor from the host cell may be related (or identical) to the essential bacterial sites known to limit the participation of parental genomes in RF replication. Double-infection experiments with wild-type phage and phage carrying an amber mutation in gene A show that the formation of gene A-specific RFII and RI is cis-limited to only the wild-type DNA. These results provide a basis at the DNA level for the known asymmetric complementation of gene A.     

An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.     

Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms.

Multiplication of the duplex, circular, phage phiX174DNA (replicative form, RF) in stage II of the replicative life cycle has been observed with a crude enzyme preparation [Eisenberg et al. (1976) Proc, Natl. Acad, Sci. USA 73, 1594-1597]. This stage has now been partially reconstituted with purified proteins and subdivided into two stages: II(+) and II(-). In stage II(+), viral (+) strand synthesis is carried out by four proteins: the phage-induced, cistron A-dependent protein, rep-dependent protein, DNA unwinding protein, and DNA polymerase III holenzyme. In stage II(-), complementary (-) strand synthesis utilizes the product of stage II(+) as template and the multiprotein system previously identified in the stage I synthesis of a complementary strand on the viral DNA template to produce RF. The multiprotein system includes DNA unwinding protein, proteins i and n, dnaB protein, dnaC protein, dnaG protein, and DNA polymerase III holoenzyme. A discussion of these two separate mechanism for synthesis of (+) and (-) strands suggests that they may account for essentially all the replicative stages in the life cycle of phiX174.     

Site-specific cleavage of DNA at 8- and 10-base-pair sequences.

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.     

Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.     

Restriction endonuclease cleavage map and location of unique features of the DNA of hepatitis B virus, subtype adw2.

DNA of hepatitis B virus (HBV) of hepatitis B surface antigen (HBsAg) subtype adw2 made fully double stranded by the virion DNA polymerase and radiolabeled either by the virion DNA polymerase reaction or by nick-translation with 32P-labeled deoxynucleoside triphosphates was used to establish a map of restriction endonuclease cleavage sites by the method double and triple enzyme digestion and to determine the relative positions of several unique physical features of this DNA. The five restriction sites for enzyme HincII, the two sites each for BamHI, Ava I, and Bgl II, and the single sites for EcoRI, Pst I, Hpa I, and Taq I were positioned relative to each other. Within this map, the single-stranded region in HBV DNA has been localized and the locations of nicks in each strand (a and b) have been determined with respect to restriction sites on the circular map. Comparison of restriction endonuclease cleavage patterns of DNAs of HBV of HBsAg subtypes adw2, ayw3, and adrq+ revealed consistent differences among subtypes and occasional differences within subtypes.     

Replication of duplex DNA of phage phi X174 reconstituted with purified enzymes.

Replication of the covalently closed duplex replicative form (RF) of phage phi X174 DNA has been achieved by coupling two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (ii) conversion of SS to nearly complete RF (RF II). In this coupled system, activated RF (gene A . RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The 11 proteins required for the two component systems were all needed in the coupled RF duplication system; no new factors were required. Single-stranded DNA binding protein was needed for RF duplication at only 4% the level needed in its stoichiometric participation in SS synthesis. In addition to RF II, more complex replicative forms appeared late in the reaction, and their possible origin is discussed.     

Infidelity of DNA synthesis associated with bypass of apurinic sites.

The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.     

Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.

The rate of reaction of double-stranded replicative form (RF) [3H]DNA of bacteriophage phiX174 with excess (+)strand DNA and (+)strand RNA was measured by standard methods of hydroxyapatite chromatography. The reactions follow pseudo-first-order kinetics and the observed rate constant for the RNA-DNA reaction differs less than 25% from that of the DNA-DNA reaction. The pseudo-first-order rate constants are close to the value predicted on the basis of the second-order rate constant measured in the renaturation of the double-stranded phiX RF [3H]DNA.