Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.     

Interruption-deficient mutants of bacteriophage T5. II. Properties of a mutant lacking a specific interruption.

An examination was made of the properties of T5HA4, a mutant of bacteriophage T5 that lacks the single-chain interruption that occurs at 7.9% from the left end of the genome. The DNAs of T5HA4 and the wild type were compared by electrophoresis in agarose gels of both single-stranded fragments produced by denaturation and duplex fragments generated by sequential treatment with exonuclease III and SI nuclease. These studies demonstrated that T5HA4 also lacks an interruption that occurs at 99.6% in wild-type DNA. The interruptions at 7.9 and 99.6% therefore occur within the 8.3% of T5 DNA that is terminally repetitious. Evidence on the location of other interruptions within the terminal repetition was also obtained. Analysis of T5HA4 with a restriction endonuclease indicated that the interruption deficiency is not due to a deletion or addition mutation. The injection of T5HA4 DNA into a host bacterium was found to occur, as with the wild type, in a two-step manner. The interruption at 7.9% is therefore not required for stopping DNA transfer after the initial 8% segment has been injected.     

Localization of the arginine tRNA gene to the D segment of T5 bacteriophage DNA. A new procedure for producing duplex DNA fragments

The tRNA genes of bacteriophage T5 are located in four clusters on the continuous heavy DNA strand (Chen, M.-J., Locker, J., and Weiss, S.B. (1976) J. Biol. Chem. 251, 536--547). Three of the four clusters are within the DNA C segment; the fourth cluster, to which only tRNAArg has been localized, maps in a 3.02 kilobase (kb) region of which 1.99 kb are at the right end of the C segment and 1.03 kb at the left end of the D segment. In order to localize the tRNAArg gene further and to define its relationship to the C-D nick, we devised a suitable method for preparing T5 DNA fragments whose ends correspond to the position of the T5 DNA nicks contained in the light DNA strand. In this method, DNA is denatured, partially renatured, and digested with low concentrations of S1 nuclease. Agarose-gel electrophoresis of these digests gives a pattern of bands which correlate in size with the pattern expected from the nicked structure of T5 DNA. Annealing of individual purified T5 [35P]tRNA species to the T5 DNA fragments transferred to nitrocellulose filters shows that tRNAArg hybridizes exclusively to the D fragment and is therefore localized to 1.03 kb at the 5' (left) end of the heavy strand of the D segment. This finding suggests that the promotor for this early gene is to the right of the C-D nick in T5 DNA; hence, the C-D nick does not coincide with this early promotor.     

Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion

Incubation of 32P-5' end-labeled DNA fragments of less than 500 bp with excess amounts of the 3' leads to 5', double strand-dependent nuclease Exonuclease III generally results in single-stranded products of slightly more than half the size of the uncleaved substrate. When such restriction fragments of known size and sequence containing the lac operator were incubated with purified lac repressor, Exonuclease III cleavage was blocked at the 3' borders of the operator on each strand. It was possible to define the DNA sequence between the two boundaries of repressor-mediated exonuclease blockade by electrophoresing the single-stranded, protected products in urea-containing polyacrylamide gels in parallel with a dimethylsulfate modification-cleavage digest of the end-labeled, uncleaved substrate. The same approach was applied to an analysis of sites of large SV40 T antigen protection in the vicinity of the origin of SV40 DNA replication. Three discrete boundaries of apparent protection were observed--one on the "late" side of the origin and two on the "early" side. These sequences may constitute the 3' borders of discrete T antigen-binding sites in the origin region. Alternatively, one or more of these blockade points may signify regions of the genome which undergo conformational changes resulting in Exonuclease III resistance due to vicinal T antigen binding.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.     

Contacts between the 5' nuclease of DNA polymerase I and its DNA substrate

The 5' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a member of an important class of prokaryotic and eukaryotic nucleases, involved in DNA replication and repair, with specificity for the junction between single-stranded and duplex DNA. We have investigated the interaction of the 5' nuclease domain with DNA substrates from the standpoint of both the protein and the DNA. Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that contacts are made to one face only of the duplex portion of the DNA substrate. Phosphodiester contacts were investigated further using DNA substrates carrying unique methylphosphonate substitutions, together with mutations in the 5' nuclease. These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3' to the cleavage site. Our results provide strong support for a DNA binding model proposed for the related exonuclease from bacteriophage T5, in which the conserved basic residues mentioned above define the two ends of a helical arch that forms part of the single-stranded DNA-binding region. The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis. In addition to binding the DNA backbone around the cleavage point, the 5' nuclease also has a binding site for one or two frayed bases at the 3' end of an upstream primer strand. In agreement with work in related systems, 5' nuclease cleavage is blocked by duplex DNA in the 5' tail, but the enzyme is quite tolerant of abasic DNA or polarity reversal within the 5' tail.     

Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments

Bacteriophage T4 RNase H belongs to a family of prokaryotic and eukaryotic nucleases that remove RNA primers from lagging strand fragments during DNA replication. Each enzyme has a flap endonuclease activity, cutting at or near the junction between single- and double-stranded DNA, and a 5'- to 3'-exonuclease, degrading both RNA.DNA and DNA.DNA duplexes. On model substrates for lagging strand synthesis, T4 RNase H functions as an exonuclease removing short oligonucleotides, rather than as an endonuclease removing longer flaps created by the advancing polymerase. The combined length of the DNA oligonucleotides released from each fragment ranges from 3 to 30 nucleotides, which corresponds to one round of processive degradation by T4 RNase H with 32 single-stranded DNA-binding protein present. Approximately 30 nucleotides are removed from each fragment during coupled leading and lagging strand synthesis with the complete T4 replication system. We conclude that the presence of 32 protein on the single-stranded DNA between lagging strand fragments guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the RNA primers, and that the difference in the relative rates of synthesis and hydrolysis ensures that there is usually only a single round of degradation during each lagging strand cycle.     

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.     

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.     

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.     

A Streptomyces glaucescens endodeoxyribonuclease which shows a strong preference for CC dinucleotide.

We have characterized a deoxyribonuclease from Streptomyces glaucescens that cleaves double-stranded DNA preferably between the dinucleotide 5'-CC-3'. The cleavage specificity was demonstrated by both analysis of the terminal nucleotides of the generated fragments and DNA sequencing of partially digested DNA. Digestion of lambda DNA with this enzyme resulted in the production of double-stranded fragments with 5' and/or 3'-protruding single-stranded tails. DNase I footprinting experiments indicated that the nuclease specifically binds to its cleavage sites on the DNA under non-catalytic conditions. The enzyme is not affected by cytosine methylation in hemimethylated DNA.     

Sequence analysis of the nicks and termini of bacteriophage T5 DNA.

Bacteriophage T5 DNA, when isolated from mature phage particles, contains several nicks in one of the two strands. The 5'-terminal nucleotides at the nicks were labeled with polynucleotide kinase and [gamma-32P]ATP, and the 3'-terminal nucleotides were labeled with Escherichia coli DNA polymerase I and [alpha-32P]dGTP. The sequences around the nicks were analyzed by partial nuclease digestion followed by homochromatography fractionation of the resulting oligonucleotides. The nicks had at least the sequence -PuOH pGpCpGpC- in common. In addition, the two 5' external termini had the first seven nucleotides in common.     

The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.     

Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity

The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

A conserved tyrosine residue aids ternary complex formation,but not catalysis,in phage T5 flap endonuclease

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.     

The chromosome of bacteriophage T5 : II. Arrangement of the single-stranded DNA fragments in the T5 and T5st(O) chromosomes

Denatured bacteriophage T5 DNA contains a large number of single-stranded DNA fragments which have been separated by agarose gel electrophoresis and classified as “major” or “minor” species on the basis of their relative abundances (Hayward & Smith, 1972). For further study of these fragments we have centrifuged denatured T5 DNA in CsCl density-gradients in the presence of poly(G). Gel electrophoretic analysis of fractions from these gradients shows that the 37.0 and 13.9 million major fragments of T5⁺ DNA and the 35.3 and 17.2 million of T5st(O) DNA are found in the high buoyant density regions. The other fragments vary in the extent of their interactions with poly(G) and a minor fragment, which has anomalous electrophoretic properties, exhibits the strongest poly(G) interaction.     

Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins

Rolling circle replication from M13 DNA circles was previously reconstituted in vitro using purified factors encoded by bacteriophage T4. The products are duplex circles with linear tails >100 kb. When T4 DNA polymerase deficient in 3' to 5' exonuclease activity was employed, electron microscopy revealed short single-stranded DNA "flaps" along the replicated tails. This marked the beginning of each Okazaki fragment, allowing an analysis of the lengths of sequential Okazaki fragments on individual replicating molecules. DNAs containing runs of Okazaki fragments of similar length were found, but most showed large length variations over runs of six or more fragments reflecting the broad population distribution.     

Properties of the 3' to 5' exonuclease associated with calf DNA polymerase delta

The 3' to 5' exonuclease of calf thymus DNA polymerase delta has properties expected of a proofreading nuclease. It digests either single-stranded DNA or the single-stranded nucleotides of a mismatched primer on a DNA template by a nonprocessive mechanism. The distribution of oligonucleotide products suggests that a significant portion of the enzyme dissociates after the removal of one nucleotide. This mechanism is expected if the substrate in vivo is an incorrect nucleotide added by the polymerase. Digestion of single-stranded DNA does not proceed to completion, producing final products six to seven nucleotides long. Digestion of a long mismatched terminus accelerates when the mismatched region is reduced to less than six nucleotides. At the point of complementation, the digestion rate is greatly reduced. These results suggest that short mismatched regions are a preferred substrate. The use of a mismatched primer-template analogue, lacking the template single strand, greatly lowers digestion efficiency at the single-stranded 3'-terminus, suggesting that the template strand is important for substrate recognition. When oligonucleotides were examined for effectiveness as exonuclease inhibitors, (dG)8 was found to be the most potent inhibitor of single-stranded DNA digestion. (dG)8 was less effective at inhibiting digestion of mismatched primer termini, again suggesting that this DNA is a preferred substrate. Overall, these results indicate that the exonuclease of DNA polymerase delta efficiently removes short mismatched DNA, a structure formed from misincorporation during DNA synthesis.