Cleavage and circularization of single-stranded DNA: a novel enzymatic activity of phi X174 A* protein.

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.     

Properties of the A and A proteins of bacteriophage G4. The origin of G4 replicative-form DNA replication

The A and A proteins of the bacteriophage G4 have been purified. The proteins have been analysed for their enzymatic activities on single-stranded and double-stranded DNA. The A protein introduces a single-stranded break at a specific place in the G4 replicative form I DNA. This cleavage site has been localized between nucleotides 506 and 507 in the viral (+) strand. The A protein binds covalently to the 5' end of the cleavage site. The A protein initiates the replication of the viral (+) DNA [Borrias, et al. (1979) Virology, 31, 288-298]; the cleavage site therefore identifies the origin of replication. The A protein cleaves viral (+) strand DNA at many different sites and also binds covalently to the 5' ends of the nick sites. The properties of both proteins strongly resemble the properties of the A and A proteins of the related and much butter analysed phage phi X174. These results indicate that the G4 and phi X174A and A proteins have comparable functions and also that both phages initiate the replicative form DNA in a similar way.     

Further characterization of the phosphate moiety of the adenovirus type 2 DNA-binding protein

The adenovirus type 2 DNA-binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single-stranded and double-stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA-binding protein into two subspecies of Mr about 45000 and 25000. The 45000-Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000-Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA-binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4--7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA-binding protein. The DNA-binding protein isolated from infected cells after a short 'pulse' of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a 'chase' period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA-binding protein, indicating that the modification of the protein is due to phosphorylation.     

Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex.

The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.     

A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase.

The polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid R1162 has been purified as a beta-galactosidase fusion protein. The hybrid protein binds specifically to a small, double-stranded DNA fragment containing the origin of transfer (oriT), and specifically cleaves oriT single-stranded DNA at the position cleaved during transfer. Only one of the two DNA strands is a substrate. A fraction of the digested DNA is resistant to lambda exonuclease digestion, indicating that some molecules have protein covalently attached at the 5' end. After prolonged incubation with fusion protein, some of the cleaved molecules are religated. In vivo, M13 phage DNA containing two, directly-repeated copies of oriT recombine in cells containing the fusion protein. The single-stranded viral DNA forms are the probable substrates for the protein, the cleaved DNA being subsequently religated to form recombinant molecules. Cleavage of the DNA might be the reverse reaction of the ligation that normally takes place after conjugative transfer of a single, linear plasmid DNA strand.     

Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.     

Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5''-dAMP.

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.     

Location of the T4 Gene 32 Protein Binding Site on Simian Virus 40 DNA

The T4 gene 32 protein, which binds to single-stranded but not duplex DNA, forms a specifically located denaturation loop in covalently closed circular simian virus 40 (SV40) DNA. Cleavage of the SV40 DNA-gene 32 protein complex with a restriction endonuclease from Hemophilus parainfluenzae shows the loop center to be at 0.46 on the SV40 DNA map. This is within one of the regions of SV40 DNA cleaved preferentially by the single-strand-specific nuclease S(1).     

Study of interaction of human replication factor A with DNA using new photoreactive analogs of dTTP

Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA. RPA is composed of three subunits with molecular masses 70 (p70), 32 (p32), and 14 kD (p14). The photoaffinity labeling method was used to study the interaction of RPA with the 3;-end of duplex DNA containing extended 5;-end of a single strand. We have synthesized dTTP analogs containing photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group attached to the 5th position of the uracil residue with linkers of variable length (9, 11, and 13 atom chains). Using these analogs and dTTP analog containing the same photoreactive residue attached to the 5th position of the uracil residue with a 4-atom linker, a number of oligonucleotide primers carrying a single photoreactive group on the 3;-end were enzymatically synthesized. Using the complex of the photoreactive primers with DNA template containing extended 19-base 5;-end, human RPA was photoaffinity modified. The primers were covalently bound to the p70 and p32 subunits of RPA and the p14 subunit was not labeled by the primers. The data are discussed considering the previously suggested model of interaction of RPA with DNA during replication.     

T4 phage gene uvsX product catalyzes homologous DNA pairing.

Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single-stranded DNA-dependent ATPase activity. It binds efficiently to single- and double-stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double-stranded DNA-protein complex was stable, but the single-stranded DNA-protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat-denatured double-stranded DNA, and assimilated linear single-stranded DNA into homologous superhelical duplexes to produce D-loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double-stranded DNA and homologous, circular single-stranded DNA, the protein catalyzed single-strand displacement in the 5' to 3' direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single-strand assimilation and single-strand displacement are briefly discussed.     

The complex of T4 bacteriophage gene 44 and 62 replication proteins forms an ATPase that is stimulated by DNA and by T4 gene 45 protein

The bacteriophage T4 genome is believed to encode all of the proteins needed for the replication of its own DNA. Included among these proteins are the "polymerase accessory proteins", the products of T4 genes 44, 62 and 45. The first two of these genes specify the synthesis of the 44/62 protein complex, which is here shown to be a DNA-dependent ATPase, hydrolyzing either ATP or dATP to the corresponding nucleoside diphosphate and releasing inorganic phosphate. This nucleotide hydrolysis is greatly stimulated by addition of the gene 45 protein and by single-stranded DNA termini. A rapid micro DNA-cellulose assay is introduced and used to measure accessory protein binding to the complex of T4 gene 32 protein and single-stranded DNA. In the presence of ATP, the 44/62 protein binds to this complex but not to naked DNA, while the 45 protein requires both the 32 protein and the 44/62 protein for detectable binding.     

Plasmid RK2 ParB Protein: Purification and Nuclease Properties

The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.     

A protein covalently bound to ColE1 DNA

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.     

Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein

The 5′ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M_r protein, linked to a small [³²P]oligonucleotide, was isolated after DNase digestion of uniformly ³²P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [³²P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5′ ends of Ad5 DNA); (3) prior treatment of the [³²P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of O-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mm-NaOH at 70 °C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad5 DNA under these conditions we were able to label the 5′ termini with ³²P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5′ mononucleotides yielded [³²P]dCMP. We conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the β-OH of a serine residue of the protein and the 5′-OH of the terminal deoxycytidine residue of the DNA.     

The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

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.     

Combinatorial selection of a single stranded DNA thioaptamer targeting TGF-beta1 protein

A phosphorothioate single-stranded DNA aptamer (thioaptamer) targeting transforming growth factor-beta1 (TGF-beta1) was isolated by in-vitro combinatorial selection. The aptamer selection procedure was designed to modify the backbone of single-stranded DNA aptamers, where 5' of both A and C are phosphorothioates, since this provides enhanced nuclease resistance as well as higher affinity than that of a phosphate counterpart. The thioaptamer selected from a combinatorial library (5x10(14) sequences) binds to TGF-beta1 protein with an affinity of 90 nM. In this report, sequence, predicted secondary structure, and binding affinity of the selected thioaptamer (T18_1_3) are presented.     

High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.     

Topoisomerase from Ustilago maydis forms a covalent complex with single-stranded DNA through a phosphodiester bond to tyrosine

Highly purified topoisomerase from Ustilago breaks single-stranded DNA, forming a complex with protein covalently bound to the DNA. Methods used to detect the complexes include a nitrocellulose filter assay, electrophoresis of the DNA-protein complex in agarose gels containing alkali, and isolation of the complex after removal of all but a small oligonucleotide fragment bound to the protein. The linkage of the Ustilago topoisomerase is to the 3' end of the broken strand of DNA. The DNA-protein complex formed is through a phosphodiester bond to tyrosine.