Single-stranded Fd DNA Research Articles

DNA synthesis catalyzed in vitro by yeast extracts.

A procedure to prepare crude extracts from single colonies of Saccharomyces cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single-stranded fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size, Isopycnic analysis of the products of ribonucleotide-initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd-replicating capacity of extracts prepared from cell-division-cycle mutants, defective in events controlling DNA initiation of elongation, showed an increased thermosensitivity in the DNA replication reaction in vitro.

Structures of fd Gene 5 Protein·Nucleic Acid Complexes: A Combined Solution Scattering and Electron Microscopy Study

Small-angle scattering and electron microscopy studies of fd gene 5 protein (g5p) and reconstituted g5p·nucleic acid complexes have been used to test models for the complexes and evaluate their uniqueness. In addition, we have obtained new information on the dependence of nucleotide type and protein/nucleotide (P/N) ratio on the structure of the complexes. Reconstituted complexes were made with single-stranded fd viral DNA (fd ssDNA), poly[d(A)] and poly[r(A)]. All complexes form similar left-handed, flexible superhelices having approximately the same diameter, but the pitch differs among these complexes. The g5p protein is a dimer in solution and the dimers associate to form a superhelical framework to which the polynucleotide is attached. The combined X-ray and neutron scattering data confirm the nucleic acid is inside the protein superhelix. A Monte Carlo integration modeling procedure applied to the scattering data was used to systematically test large numbers of possible models for each complex, and previously proposed models based on parameters obtained from electron microscopy were found to be essentially correct and unique. The data on the complexes with differentP/νratios showed that mass per unit length values decreased while the rise per dimer and pitch of the superhelix increased for g5p·fd-ssDNA complexes with decreasingP/Nratios.

Nuclear matrix-bound DNA primase. Elucidation of an RNA priming system in nuclear matrix isolated from regenerating rat liver.

Recent findings in purified systems demonstrate the universality of DNA polymerase-primase complexes which may function in the priming and continuation of eucaryotic DNA replication. In this report we characterize an in vitro, nuclear matrix-associated, priming and continuation system that can utilize either endogenous matrix-bound DNA or exogenous single-stranded DNA as template. 30-40% of total nuclear DNA primase activity was recovered in association with the isolated nuclear matrix fraction from regenerating rat liver. Matrix-bound primase catalyzed the alpha-amanitin, actinomycin D-resistant synthesis of oligonucleotide chains of 8-50 nucleotides on the endogenous template. At least a portion of the RNA primers were continued by DNA polymerase alpha with deoxynucleoside triphosphate incorporation up to 300-600 nucleotides. Nearest neighbor analysis revealed ribodeoxynucleotide covalent linkages in these RNA-DNA chains. The matrix-bound primase preferred single-stranded fd DNA as exogenous template over synthetic homopolymers and was strictly dependent on the presence of ribonucleoside triphosphates. Appropriate subfractionation revealed that the matrix-bound primase activity is exclusively localized in the nuclear matrix interior. The ability of primase and DNA polymerase to synthesize covalently linked RNA-DNA products demonstrates the potentially useful role of the nuclear matrix in vitro system for elucidating the organizational and functional properties of the eucaryotic replication apparatus in the cell nucleus.

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY.

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.

The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration.

Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.

Electron microscopic visualization of RecA-DNA filaments: Evidence for a cyclic extension of duplex DNA

As visualized by electron microscopy, RecA protein binds in a highly cooperative manner to single-stranded fd DNA in solutions of 0.01 M Tris (pH 7.5). The resulting nucleoprotein filament loops are 1.25 μm in length, have a fiber diameter of 12 nm and show an indication of a 4.5 nm repeat along the axis of the compact fibers. RecA binds to linear duplex fd DNA in solutions of 0.01 M Tris (pH 7.5) to yield chains of beads which, in the presence of Mg 2+ and ATP, coalesce into smooth filaments with a length of 1.9 μm (the length of protein-free fd duplex DNA) and have a fiber diameter of 12 nm. In solutions containing Mg 2+ and ATP-γ-S, however, RecA binds to duplex DNA in a highly cooperative manner to yield rigid filaments 3.0 μm in length. These filaments are 12 nm in diameter and show a very clear 7.5 nm axial repeat. This extension of DNA to 150% of its usual length in the apparent absence of any single-stranded components suggests that the DNA helix must also be highly unwound and provides new insights into the mode of RecA action.

Primary and secondary structure specificity of the cleavage of 'single-stranded' DNA by endonuclease Hinf I.

The interaction of endonuclease Hinf I with single-stranded fd DNA was examined. The sizes of the cleavage products indicate that the enzyme cuts this substrate at the same sequences as double-stranded DNA (GANTC). To determine whether or not the recognition sites in single-stranded DNA have to be present in double-stranded form in order to be cleaved, DNA fragments containing complementary or non-complementary Hinf I sequences were prepared and treated as substrates. The results suggest that completely base-paired recognition sites are necessary for cleavage. Sequences surrounding the Hinf I pentanucleotides significantly modulate the reaction rates.

Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template.

We have recently developed an in vitro DNA synthesis system in which a synthetic heptaribonucleotide pairs with a unique site on a single-stranded fd DNA molecule and thereby primes the growth of new DNA strands from this single point (Huang, C.-C., and Hearst, J. E. (1980) Anal. Biochem. 103, 127-139). In this report, we use this system to investigate the mechanism by which various bacteriophage T4 DNA replication proteins stimulate the T4 DNA polymerase. We find that with the "polymerase accessory proteins" present (the T4 gene 44/62 and 45 proteins), the DNA polymerase proceeds rather rapidly through the occasional hairpin helices which otherwise interrupt the progress of this enzyme along single-stranded DNA templates. By using a potent inhibitor of the 44/62 ATPase, ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), we have obtained data which suggest that ATP hydrolysis is required for the formation of a polymerase accessory protein-DNA template complex, and that this complex then persists, serving as a sliding clamp which greatly increases the strength of binding between a T4 DNA polymerase molecule and its 3'OH primer template end. The progress of the T4 DNA polymerase though hairpin helices in the DNA template is also stimulated by addition of the T4 helix-destabilizing protein (gene 32 protein). The effect of the 44/62 and 45 proteins is independent of the effect of the 32 protein in this assay, and the rate of polymerase travel over the strongest hairpin helices is increased more than 40-fold in the presence of these four additional proteins.

Pauses at positions of secondary structure during in vitro replication of single-stranded fd Bacteriophage DNA by T4 DNA polymerase

Since bacteriophage T4 DNA polymerase is unable to use duplex DNA molecules as templates ( B. Alberts, J. Barry, M. Brittner, M. Davies, H. Hama-Inaba, C. C. Liu, D. Mace, L. Moran, C. F. Morris, J. Piperno, and N. Sinha, 1977, in Nucleic Acids and Protein Recognition, Vogel, H. J., ed., pp. 31–63, Academic Press, New York), a technique involving synchronous and uniquely primed synthesis of DNA on the single-stranded fd DNA by the T4 DNA polymerase has been developed to probe regions exhibiting secondary structure on this genome. As the polymerase proceeds, the template secondary structure acts as a kinetic barrier to delay the continuous chain extension catalyzed by this enzyme. These kinetic pause sites can be mapped by denaturing agarose gel electrophoresis of replication intermediates and used to generate a secondary structure map. Using this method, we are able to establish a list including at least seven plausible stable helical regions in fd DNA. Two of the most stable secondary structures have been mapped near fd sequence positions 3350 and 5650, respectively. The latter has been reported to be the region where fd DNA replication begins ( C. P. Gray, R. Sommer, C. Polke, E. Beck, and H. Schaller, 1978, Proc. Nat. Acad. Sci. USA, 75, 50–53). However, the biological function associated with the former has yet to be investigated. Following a two-state model, we estimate the first-order rate constant for progression through the duplex regions near position 5650 in fd DNA to be about 0.042 min −1 for fd DNA synthesis by the T4 DNA polymerase under our reaction conditions. A 7.5-fold increase in this rate constant is obtained upon the addition of the T4 DNA helix destabilizing protein (i.e., gene 32 protein). The general pattern of our secondary structure map agrees well with a computer search for duplex regions on the fd genome. Both the stability and the size of a stable secondary structure at a particular position on the fd template determine the time that the newly made DNA molecules spend at that site. A structure with a stem of less than 8 base pairs does not interrupt significantly the procession of the T4 DNA polymerase during the process of fd DNA synthesis.

A specific DNA orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.

Ionic strength perturbation kinetics of gene 32 protein dissociation from its complex with single-stranded DNA

Equilibrium and kinetic studies of the interaction of gene 32 protein of T4 phage with single-stranded fd DNA were performed monitoring the changes in protein fluorescence. From the fluorescence titrations, it was estimated that a monomer of gene 32 protein covered six nucleotide bases on the DNA and the lower limit for the apparent association constant was 1.9 x 10(8) M-1 with a cooperative parameter of 10(3) in 0.1 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (pH 7) at 25 degrees C. When an ionic strength jump was applied to the gene 32 protein-fd DNA complex using a stopped-flow apparatus, the complex underwent a dissociation into its individual components accompanied by an increase in protein fluorescence. The kinetics of the dissociation are not consistent with a single first-order process. The data, however, can be analyzed in terms of a model in which gene 32 protein molecules release cooperatively starting from either one or both ends of a cluster of proteins bound to fd DNA. This type of dissociation of gene 32 protein from single-stranded DNA is very efficient and has interesting implications: it could provide a way to facilitate a rapid "zippering" of the two complementary DNA strands during DNA replication and genetic recombination.

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using (3)H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.Isolated nuclei were incubated with (3)H-labeled single-stranded bacteriophage fd DNA. Poly-l-lysine or DEAE-dextran at 0.1 and 1 mug/ml stimulated DNA binding. On the other hand, poly-l-lysine at 1 and 5 mug/ml increased DNA uptake but DEAE-dextran did not. Ten to 20 mm Ca and Mg ions were required for DNA uptake. EDTA (1-20 mm) did not differ from the control or the low levels of Ca or Mg ions. These observations on single-stranded fd DNA differed from those obtained with double-stranded DNA of Salmonella typhimurium. Kinetics of single-stranded DNA binding and uptake also deviated from that of double-stranded DNA.Analyses of nuclei-bound DNA by sucrose density gradient and CsCI density gradient centrifugation revealed extensive DNA degradation during a 45-minute incubation period. Poly-l-lysine protected against rapid degradation of bound DNA. DEAE-dextran enhanced DNA binding, but bound DNA was cleaved into much smaller polymers than those detected in control experiments or in the presence of poly-l-lysine. Sucrose density gradient and CsCI density gradient centrifugation analyses on DNA taken up by nuclei also showed extensive DNA degradation. Poly-l-lysine slightly inhibited DNA degradation, but DEAE-dextran appeared to enhance degradation of incorporated DNA. Moreover, nonbound DNA in the incubation medium was completely degraded within a 20-minute incubation period.

Photochemical cross-linking of DNA-RNA helices by psoralen derivatives

Bacteriophage fd DNA-RNA hybrid has been prepared in vitro by transcribing single-stranded fd DNA with Escherichia coli RNA polymerase. Most of the newly synthesized RNA was found to be associated with DNA on the basis of buoyant density studies in Cs 2SO 4 density gradients. First, the buoyant density of the complex increases with the extent of transcription. Secondly, the native complex is resistant to pancreatic RNAase digestion in 0.2 m-NaCl (37 °C, 30 min), whereas RNA of the heat-denatured complex can be hydrolyzed by the enzyme under the same conditions. The ability of psoralens to covalently cross-link DNA-RNA double helices has been tested by irradiating either 3H-labeled fd DNA- 32P-labeled RNA hybrid or unlabeled fd DNA- 32P-labeled RNA hybrid with long wavelength ultraviolet light in the presence of one of the three psoralen derivatives: 4′-aminomethyl-4,5′,8-trimethylpsoralen hydrochloride, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen, or 4,5′,8-trimethylpsoralen. While the RNA and DNA strands of the native complex separate irreversibly after denaturation with heat or heat plus urea, the psoralen + u.v.-treated hybrids undergo a reversible helix-coil transition as shown by equilibrium Cs 2SO 4 density gradient ultracentrifugation, gel electrophoresis, and RNAase digestion experiments. These results indicate that covalent bridges form between the RNA and DNA strands after the photochemical reaction of the hybrid with AMT, HMT or TMP. The photochemically cross-linked DNA-RNA complexes have been denatured completely using the 37 °C (formaldehyde + formamide) technique (Shen & Hearst, 1977 b) and spread for electron microscopy to examine the interstrand cross-links directly. The average number of cross-links was calculated for these looped and tailed hybrid molecules and compared to that of TMP cross-linked and denatured double-stranded fd DNA restriction fragments. From the denaturation microscopy studies, it has been concluded that: (1) under the conditions of low cross-linking (9 μ m-base-pairs, 0.045 μ m-psoralen molecules, 10 m m-Tris · HCl, 1 m m-EDTA, 10 m m-NaCl, pH 8.0, 15 °C), the AMT, HMT and TMP hybrids have 2.0, 2.2 and 2.2 cross-links per molecule, respectively; (2) as the salt is increased to 200 m m-NaCl, the average number of the cross-links decreases to 0.7, 0.9 and 1.5; (3) TMP cross-links DNA helices more efficiently than DNA-RNA helices by factors of approximately 4 and 6 in 10 m m and 200 m m-NaCl, respectively; (4) the photochemical cross-linking reaction can be driven by higher concentrations of psoralens. With an initial concentration of 0.45 μ m-psoralen, the distance between adjacent cross-links decreases appreciably and many apparent double-stranded segments are observed on the denatured molecules. After the psoralen concentration is increased to 9 μ m, loops or tails are seen on only a few of the molecules, indicating that the DNA-RNA hybrid becomes densely cross-linked. The photochemical cross-linking reaction of DNA-RNA hybrid with psoralens should become a powerful tool in studying chromosome structure and genetic activities involving “transient” DNA-RNA hybrids.

Interaction of DNA with DNA binding proteins III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.

Interaction of DNA with DNA binding proteins II. Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD

Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA. Electron microscopy reveals granular substructures in the complexes formed with protein HD. In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone. Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome. The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes.

Knotted single-stranded DNA rings: A novel topological isomer of circular single-stranded DNA formed by treatment with Escherichia coli ω protein

Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.

Psoralen-crosslinked secondary structure map of single-stranded virus DNA.

The photochemical crosslinking of DNA by 4,5',8-trimethylpsoralen (trioxsalen) has been used to freeze the secondary structures of single-stranded DNA molecules of bacteriophage fd at different ionic strengths. These secondary structures (hairpins or looped hairpins) have been visualized in the electron microscope. Most of the single-stranded circular fd DNA molecules show only one hairpin after irradiation at 15 degrees in 20 mM NaCl in the presence of trioxsalen. As the ionic strength is increased, more hairpins appear on the DNA molecules. To map these secondary structures, double-stranded supercoiled fd DNA (RFI) was cleaved with the restriction enzyme HindII, which makes only one cut on each RFI molecule. After denaturation and crosslinking of the linear single-stranded fd DNA (a mixture of viral and complementary strands), all the hairpins have been mapped on the DNA molecule with respect to this HindII site. The results show that these hairpins occur at specific sites. The most stable hairpin has been assigned to the position where the initiation site for the conversion of single-stranded fd DNA to the double-stranded covalently closed form has been mapped. The remaining hairpins map in or near regions corresponding to in vitro promoter sites on the fd DNA.

Three DNA polymerases of rat ascites hepatoma cells: Properties of the enzymes and effect of RNA synthesis on the reactions

Three different DNA polymerases have been isolated from rat ascites hepatoma cells [1–3]. The molecular weight of a DNA polymerase (polymerase C) purified from the soluble fraction of the cells was estimated to be 142 000 by sedimentation on a sucrose gradient, while the molecular weights of two DNA polymerases (polymerase P-1 and P-2) purified from nuclear membrane-chromatin fraction were estimated to be 117 000 and 44 000, respectively, by the same method. Under certain conditions, the poly (dT) strand of poly[(dA) · (dT)] was copied well by the polymerases, especially by the nuclear polymerases. Poly (dC) was good template for the high molecular weight DNA polymerases C and P-1, but poly(dT) and poly(dA) were not effective templates. By addition of complementary oligoribonucleotides, the single-stranded deoxypolymers were copied by the high molecular weight polymerases C and P-1. When single-stranded fd phage DNA was used as template, the polymerization reactions by the high molecular weight polymerases were stimulated by the concomitant synthesis of RNA. This indicates that the oligoribonucleotide acts as a primer in these reactions.

Molecular weight of single-stranded fd bacteriophage DNA. High speed equilibrium sedimentation and light scattering measurements.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMolecular weight of single-stranded fd bacteriophage DNA. High speed equilibrium sedimentation and light scattering measurementsSteven A. Berkowitz and Loren A. DayCite this: Biochemistry 1974, 13, 23, 4825–4831Publication Date (Print):November 1, 1974Publication History Published online1 May 2002Published inissue 1 November 1974https://doi.org/10.1021/bi00720a022RIGHTS & PERMISSIONSArticle Views105Altmetric-Citations56LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (850 KB) Get e-Alerts Get e-Alerts

Transcriptional Role in Deoxyribonucleic Acid Replication: NATURE OF PRIMER FUNCTION OF NEWLY SYNTHESIZED RIBONUCLEIC ACID IN VITRO

Abstract DNA synthesis in an in vitro system proceeds by a covalent chain extension of the newly generated primers in the presence of Mg2+, Escherichia coli DNA polymerase I or its large fragment, ribo- and deoxyribonucleoside triphosphates, RNA polymerase, and either single-stranded fd DNA or d(T)1000 as template. The synthesis is strongly influenced by the number of RNA chains and the kinetics of polymerization is nonlinear, showing a brief lag followed by a period of rapid synthesis before reaching a saturation level. However, a linear kinetics is observed when the number of new chain initiation is controlled (i.e. rifampicin added 1 to 5 min after RNA synthesis). A degradation of ribo-strands is observed during DNA synthesis. When d(T)1000 fully transcribed with RNA polymerase (1:1 hybrid) is employed as template-primer, both the whole enzyme and the large fragment of DNA polymerase I catalyze incorporation of dAMP residues. During the initial synthetic period there is a mole for mole degradation of ribo-strands by the whole enzyme. With large fragment DNA chain growth proceeds by a process of of strand displacement. In the initial synthetic period the 5'→3' exonuclease activity of DNA polymerase I attacks the 5' ends of the ribostrands endonucleolytically liberating 5'-triphosphate-terminated di- or oligonucleotides. Subsequent action of the nuclease activity proceeds exonucleolytically liberating 5'-mononucleotides as the DNA chain growth proceeds. The results suggest that the 5'→3' exonuclease activity of DNA polymerase is capable of removing RNA primers from the final product of replication in vivo.