Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase

RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate).     

Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts

It is shown that circular PM2 DNA with two gaps of 13 nucleotides per molecule is degraded by purified recBC enzyme from Escherichia coli to acid-soluble material at a rate which is less than one tenth of the rate of solubilization of linear duplex DNA. Increasing the gap length in the circular DNA to 40-650 nucleotides does not affect the breakdown of the molecules by the recBC enzyme, nor does it change the proportions of the products formed (acid-soluble material, acid-insoluble fragments and non-degraded molecules). On the other hand, terminal gaps in linear duplex DNA produced by limited digestion with either exonuclease III or lambda exonuclease significantly reduce the rate of the degradation by the recBC enzyme, particularly when the gaps exceed 100 nucleotides. The results suggest that the recBC enzyme does not cleave gaps in circular DNA at random positions, but possibly at the junction between single-stranded and duplex DNA or close to it. The degradation of gapped circular DNA by purified recBC enzyme was used to search for an inhibitor of the recBC enzyme in extracts from ultraviolet-irradiated cells. No such inhibitor has been observed but rather a weak stimulatory factor for the solubilization of gapped circular DNA by the recBC enzyme. Thus, the experimental system appears not to be suited as a test in vitro for an ultraviolet-induced inhibitor of the recBC enzyme which has been postulated to be produced in recA+ lexA+ cells of E. coli after ultraviolet irradiation.     

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.     

Differential down-regulation of protein kinase C selectively affects IgE-dependent exocytosis and inositol trisphosphate formation.

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.     

Postinfection control by bacteriophage T4 of Escherichia coli recBC nuclease activity.

Infection by bacteriophage T4 has previously been shown to cause a rapid inhibition of the host recBC DNase, an ATP-dependent DNase that is required for genetic recombination in Escherichia coli. We report here the partial purification of a protein ("T4 rec inhibitor") from extracts of T4-infected cells and some characteristics of the in vitro inhibition reaction with purified inhibitor and recBC nuclease. This inhibitory activity could not be purified from extracts of uninfected E. coli. Both the ATP-dependent exonuclease and DNA-dependent ATPase activities of recBC DNase are inhibited by T4 rec inhibitor. Experiments suggest that the inhibitor interacts with the nuclease in a stoichiometric manner. The biological significance of this inhibition is discussed with respect to control reactions in phage-infected cells.     

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7.

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.     

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells.

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.     

Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene.

The gam gene of bacteriophage Mu encodes a protein which protects linear double stranded DNA from exonuclease degradation in vitro and in vivo. We purified the Mu gam gene product to apparent homogeneity from cells in which it is over-produced from a plasmid clone. The purified protein is a dimer of identical subunits of 18.9 kd. It can aggregate DNA into large, rapidly sedimenting complexes and is a potent exonuclease inhibitor when bound to DNA. The N-terminal amino acid sequence of the purified protein was determined by automated degradation and the nucleotide sequence of the Mu gam gene is presented to accurately map its position in the Mu genome.     

Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S. cerevisiae.

Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.     

Deoxyribonuclease I generates single-stranded gaps in chromatin deoxyribonucleic acid

Production of 10-base multiple DNA ladder fragments during DNase I digestion of chromatin is explained by a model which does not involve site-specific nicking by the DNase I. This model was tested because it explains why 10-base (actually 10.4 base) multiple-related fragments are paradoxically generated by both endonucleolytic (DNase I) and exonucleolytic (exonuclease III) mechanisms. This new model also explains the phenomenon of substantial single-stranded DNA production during DNase I digestion of chromatin. The latter phenomenon has been widely observed but is not explained by previous models. The single-stranded gap model to be presented makes testable predictions. Primarily, these are that DNase I produces single-stranded gaps in chromatin DNA and that the termini of 10-base multiple ladder fragments are separated by single-stranded gaps. Single-stranded gap production by DNase I was confirmed by a number of methods. Sensitivity of ladder band components (from DNase I but not staphylococcal nuclease digests) to S1 nuclease suggested that the ladder fragments themselves may compose a significant portion of these gaps. Separation of ladder fragment termini by single-stranded gaps was verified by demonstrating both resistance to the nick-specific NAD+-dependent ligase and sensitivity to T4 ligase which can ligate across gaps. Many single-stranded gaps, occurring both individually and clusters, were observed by electron microscopy using either cytochrome c labeling (where the gaps) are thinner than duplex) or gene 32 protein labeling (gaps thicker than duplex). Gap sizes were estimated by protecting them with gene 32 protein and digesting away unprotected duplexes. By this method, gap sizes fall into a ladder distribution (from 10 or 20 bases up to 120 bases), which, at least in the region of the shorter sizes, clearly indicates the sizes of single-stranded gaps formed in chromatin by DNase I.     

DNase I - DNA interaction alters DNA and protein conformations.

Human DNase I is an endonuclease that catalyzes the hydrolysis of double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions in the presence of divalent Mg and Ca cations. It binds to the minor groove and the backbone phosphate group and has no contact with the major groove of the right-handed DNA duplex. The aim of this study was to examine the effects of DNase I - DNA complexation on DNA and protein conformations.We monitored the interaction of DNA with DNase I under physiological conditions in the absence of Mg2+, with a constant DNA concentration (12.5 mmol/L; phosphate) and various protein concentrations (10-250 micromol/L). We used Fourier transfrom infrared, UV-visible, and circular dichroism spectroscopic methods to determine the protein binding mode, binding constant, and effects of polynucleotide-enzyme interactions on both DNA and protein conformations. Structural analyses showed major DNase-PO2 binding and minor groove interaction, with an overall binding constant, K, of 5.7 x 10(5) +/- 0.78 x 10(5) (mol/L)-1. We found that the DNase I - DNA interaction altered protein secondary structure, with a major reduction in alpha helix and an increase in beta sheet and random structures, and that a partial B-to-A DNA conformational change occurred. No DNA digestion was observed upon protein-DNA complexation.     

Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus

Reaction intermediates formed during the degradation of linear PM2, T5, and λ DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn²⁺ (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or λ DNA, which do not contain nicks. In the presence of Mg²⁺, when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length λ DNA, EcoRI restriction fragments of λ DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3′ → 5′ direction. If this is correct, and since only 5′-monophosphate nucleosides are produced, then HSV DNase should “activate” DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg²⁺ or Mn²⁺, activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3′ → 5′ direction as well as in the 5′ → 3′ direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner.     

Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity.     

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.     

Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta

Pur alpha is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Pur alpha unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Pur alpha are essential for both ss- and duplex DNA binding. Pur alpha binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Pur alpha since removal of Pur alpha in the gel eliminates the series and since Pur alpha binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Pur alpha binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Pur beta lacking the Pur alpha C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Pur alpha can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Pur alpha.     

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.     

Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA

The avian retrovirus pp32 protein possesses DNA endonuclease activity and unique DNA binding properties. An improved purification procedure was developed for pp32, resulting in a severalfold increase in the yield of this virion protein. By use of the nitrocellulose filter binding assay, the protein retains approximately 2-fold more supercoiled (form I) DNA molecules than equivalent linear duplex DNA molecules. Single-stranded DNA is only slightly preferred over double-stranded DNA for pp32 binding. The pp32 DNA binding sites on form I pBR322 DNA which contained an insert of avian retrovirus long terminal repeat (LTR) DNA were determined. A preformed protein-DNA complex was digested with one of several different multicut restriction enzymes and filtered through nitrocellulose filters. Fragments containing viral LTR DNA sequences and plasmid DNA containing promoter sequences for the ampicillin and tetracycline genes, sequences for the "left-end" inverted repeat of transposon 3, and sequences encompassing the carboxyl terminus of the beta-lactamase gene were preferentially retained on the filter by pp32. Partial mapping of pp32 DNA binding sites on LTR DNA was accomplished by generation of deletions in LTR DNA sequences. The pp32 protein preferentially bound viral DNA fragments which contain the viral promoter (TATTTAA) and the adjacent "R" repeat sequences. Computer analysis revealed that three of the four plasmid DNA fragments retained by pp32 contained LTR DNA promoter-like sequences (one mismatch only) which were part of statistically significant and thermodynamically stable hairpin structures.     

Mechanism of DNA binding and localized strand separation by Purα and comparison with Pur family member,Purβ

Purα is a single-stranded (ss) DNA- and RNA-binding protein with three conserved signature repeats that have a specific affinity for guanosine-rich motifs. Purα unwinds a double-stranded oligonucleotide containing purine-rich repeats by maintaining contact with the purine-rich strand and displacing the pyrimidine-rich strand. Mutational analysis indicates that arginine and aromatic residues in the repeat region of Purα are essential for both ss- and duplex DNA binding. Purα binds either linearized or supercoiled plasmid DNA, generating a series of regularly spaced bands in agarose gels. This series is likely due to localized unwinding by quanta of Purα since removal of Purα in the gel eliminates the series and since Purα binding increases the sensitivity of plasmids to reaction with potassium permanganate, a reaction specific for unwound regions. Purα binding to linear duplex DNA creates binding sites for the phage T4 gp32 protein, an ss-DNA binding protein that does not itself bind linearized DNA. In contrast, Purβ lacking the Purα C-terminal region binds supercoiled DNA but not linearized DNA. Similarly, a C-terminal deletion of Purα can bind supercoiled pMYC7 plasmid, but cannot bind the same linear duplex DNA segment. Therefore, access to linear DNA initially requires C-terminal sequences of Purα.     

Differential Thermolability of Exonuclease and Endonuclease Activities of the recBC Nuclease Isolated from Thermosensitive recB and recC Mutants

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.     

DNase I-tRNA interaction

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase-RNA interaction was observed, less is known about the protein-RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I-tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg2+, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 microM to 250 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide-enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase-PO2 and minor groove interactions with overall binding constant of K = 2.1 (+/-0.7) x 10(4) M(-1). The DNase I-tRNA interaction alters protein secondary structure with major reduction of the alpha-helix, and increases the random coil, beta-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein-tRNA complexes.