Mechanism of Replication of φX174 Single-Stranded DNA IX. Requirement for the Escherichia coli dnaG Protein

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.     

Specificity of the S1 nuclease from Aspergillus oryzae.

Conditions are described for digesting single-stranded DNA by S1 nuclease without introducing breaks in double-stranded DNA. The enzyme is inhibited by low concentrations of various compounds of phosphate. Under certain conditions S1 nuclease cleaves the strand opposite a nick in bacteriophage T5 DNA; under other conditions, the enzyme cleaves a loop in one strand of heteroduplex lambdaDNA while leaving the opposite strand intact. S1 nuclease makes many single strand breaks in ultraviolet-irradiated duplex lambdaDNA. Superhelical DNA of phiX174 (Form I) is converted first to a relaxed circular molecule (Form II), and then to a linear molecule (Form III) by cleavage at one site per molecule. Since the cleavage occurs at many sites in the population of molecules, the partially single-stranded regions in phiX174 superhelical DNA are not determined by specific nucleotide sequences.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.     

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.     

Purification of the rep protein of Escherichia coli. An ATPase which separates duplex DNA strands in advance of replication.

The product of the rep gene of Escherichia coli catalytically separates phiX174 duplex DNA strands in advance of their replication, utilizing ATP in the process (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). The enzyme has now been purified to near-homogeneity. Relatively large quantities were obtained from ColE1-plasmid-containing cells in which the enzyme level was 7 to 10 times above wild type. The assay for rep protein was based on its essential role, with phage-induced cistron A protein, in enzymatic synthesis of phage phiX174 (+) strands, using duplex circular DNA as template. The protein exhibits a molecular weight of 65,000 under denaturing and reducing conditions. The turnover number of the enzyme is approximately 6800 ATP molecules/min in strand separation as measured by extent of replication, or in an uncoupled reaction using single-stranded DNA effector.     

Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication.

Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.     

Degradation of the viral strand of phiX174 parental replicative-form DNA in a rep- host.

A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled phiX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 mug/ml, a concentration sufficient to block RF leads to SS (single strand) synthesis but not RF leads to RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150 mug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand.     

Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.     

Synthesis and properties of a novel bridged nucleic acid analogue, 5'-amino-3',5'-BNA

An oligonucleotide P3'-->N5' phosphoramidate (5'-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3'-->N5' phosphoramidate linkage and a constrained sugar moiety, 5'-amino-3'-C,5'-N-methylene bridged nucleic acid (5'-amino-3',5'-BNA). The binding affinity of the 5'-amino-3',5'-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5'-amino-3',5'-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5-amino-DNA.     

The A* protein of phi X174 is an inhibitor of DNA replication

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.     

Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA.When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174.These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.     

Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli.

A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.     

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.     

Specific Fragments of φX174 Deoxyribonucleic Acid Produced by a Restriction Enzyme from Haemophilus aegyptius, Endonuclease Z

A restriction-like enzyme has been purified from Haemophilus aegyptius. This nuclease, endonuclease Z, produces a rapid decrease in the viscosity of native calf thymus and H. influenzae deoxyribonucleic acids (DNA), but does not degrade homologous DNA. The specificity of endonuclease Z is different from that of the similar endonuclease isolated from H. influenzae (endonuclease R). The purified enzyme cleaves the double-stranded replicative form DNA of bacteriophage phiX174 (phiX174 RF DNA) into at least 11 specific limit fragments whose molecular sizes have been estimated by gel electrophoresis. The position of these fragments with respect to the genetic map of phiX174 can be determined by using the genetic assay for small fragments of phiX174 DNA.     

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis

Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.