Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.     

Isolation and comparison of two molecular species of the BAL 31 nuclease from Alteromonas espejiana with distinct kinetic properties

The extracellular nuclease from Alteromonas espejiana sp. BAL 31 can be isolated as two distinct proteins, the "fast" (F) and "slow" (S) species, both of which have been purified to homogeneity. The F and S species of the nuclease have molecular weights, respectively, of 109 X 10(3) and 85 X 10(3), and both are single polypeptide chains with an isoelectric pH near 4.2. Both species catalyze the degradation of single-stranded and linear duplex DNAs to 5'-mononucleotides. The degradation of linear duplex DNA occurs through a terminally directed hydrolysis mechanism that results in the removal of nucleotides from both the 3' and 5' ends. Apparent Michaelis constants (Km) have been obtained for the exonuclease activities of both species and for the activity against single-stranded DNA of the S species. The Km for the hydrolysis of single-stranded DNA catalyzed by the F species has not been obtained because the reaction velocity was maximal even at the lowest substrate concentrations accessible in the photometric assay. The ratio of the turnover numbers for the exonuclease activities of the two species indicates that the F species will shorten linear duplex DNA at a rate 27 +/- 5 (S.D.) times faster than an equimolar concentration of the S species in the limit of high substrate concentration, while the corresponding ratio for the activities against single-stranded DNA (1.2 +/- 0.1) shows that the two species are similar with respect to hydrolysis of this substrate. In the limit of high substrate concentrations, the F and S species break phosphodiester bonds in single-stranded DNA at rates 1.3 +/- 0.3 and 33 +/- 2 times those for the exonucleolytic degradation of linear duplex DNA, respectively. It has not been established whether the two species are physically related.     

Specific limited cleavage of bihelical deoxyribonucleic acid by wheat seedling nuclease.

Wheat seedling nuclease catalyzes the hydrolysis of intact, bihelical viral DNA or high molecular weight, native Escherichia coli DNA to produce limit polymers which are resistant to further hydrolysis by additional enzyme. These limit products are double-stranded polymers free of single strand interruptions and are terminated at their 5' ends with equal amounts of either deoxycytidylate or deoxyguanylate residues. The average size of the duplex limit products, as determined by (a) alkaline and neutral sucrose gradient sedimentation, (b) viscometric determination of molecular weight, and (c) 5'-end labeling, varies from 2 to 4 times 10-6 depending on the source of the DNA. The involvement of regions rich in adenine-thymine base pairs at the sites of cleavage of the DNA molecule is suggested by the following experimental results: (a) the copolymeric duplex, poly(dA-dt) is hydrolyzed at a rate comparable to that found for denatured calf thymus DNA, a rate which is several orders of magnitude faster than that at which native calf thymus DNA is hydrolyzed; (b) lambda DNA, which contains an adenine-thymine-rich region near its center, is rapidly cleaved to yield two fragments of similar size; (c) the rate of hydrolysis of native DNA is increased approximately 14-fold by increasing the reaction temperature from 20 degrees to 30 degrees.     

Terminally directed hydrolysis of duplex ribonucleic acid catalyzed by a species of the BAL 31 nuclease from Alteromonas espejiana

The extracellular nuclease activities of Alteromonas espejiana sp. BAL 31 are mediated by at least two distinct protein species that differ in molecular weights and catalytic properties. The two species that have been purified to homogeneity and characterized, the "fast" (F) and "slow" (S) enzymes, both possess an exonuclease activity that shortens both strands of duplex DNA, with the F nuclease displaying a much greater (approximately 19-fold) turnover number for this degradation than the S species. In the present article, it is shown that the F species also mediates the terminally directed hydrolysis of a linear duplex RNA, gradually shortening molecules of this substrate through a mechanism that results in the removal of nucleotides from both the 3' and the 5' ends. This degradation proceeds with very infrequent introduction of scissions away from the termini as demonstrated by gel electrophoretic examination of the products of partial degradation, both in duplex form and after denaturation by reaction with CH3HgOH, and by electron microscopic characterization of duplex partially degraded molecules. The apparent Michaelis constant and turnover number have been determined. At equimolar enzyme concentrations in the limit of high substrate concentration, the F nuclease will degrade duplex RNA at a rate 0.021 +/- 0.010 (S.D.) times that for a duplex DNA of comparable guanine + cytosine content. The S species, by contrast, shows very little activity against the duplex RNA substrate relative to that of the F enzyme.     

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.     

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.     

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.     

Binding and conformational analysis of phosphoramidate-restriction enzyme interactions

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). Under metal-free conditions, the enzyme exhibited a remarkable approximately 50-fold greater affinity for the modified oligonucleotide relative to the native substrate (5 vs 240 nM). While (31)P NMR spectra indicate increased chemical shift dispersion in the free phosphoramidate duplex, the spectrum of the enzyme-bound duplex is similar to that of the native duplex. (1)H-(15)N HSQC analysis indicates that enzyme conformations in the presence of these oligonucleotides are also comparable. The tight binding of the phosphoramidate duplex under metal-free conditions and its resistance to cleavage are attributed to local conformational adjustments propagating from the O-->N substitution.     

The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis

Formation of D-loops during the exchange of strands between a circular single-stranded DNA and a completely homologous linear duplex proceeds optimally when the duplex DNA is added to the complex of recA protein and single-stranded DNA formed in the presence of single-stranded DNA-binding protein and ATP. D-loops are undetectable when 200 microM adenosine 5'-O-(thiotriphosphate) is substituted for ATP. D-loops can be formed in the presence of adenosine 5'-O-(thiotriphosphate) if recA protein is the last component added to the reaction. However, these D-loops, which depend upon homologous sequences, are unstable upon deproteinization and are formed to a more limited extent than the structures formed with ATP. This finding indicates that D-loops formed under these conditions may be largely nonintertwined paranemic structures rather than plectonemic structures in which two of the strands are interwoven. When adenosine 5'-O-(thiotriphosphate) is added to an ongoing reaction containing ATP, formation of plectonemic structures and ATP hydrolysis is inhibited to an equivalent extent. We, therefore, conclude that ATP hydrolysis is required for the formation of plectonemic structures.     

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5'' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.     

Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.

We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.     

Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors

ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.     

Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities.

Purified DNA polymerase III has two distinct exonuclease activities: one initiates hydrolsis at the 3 termini, and the other at the 5 termini of single-stranded DNA. Both exonucleases have the same relative mobility on polyacrylamide gels as the polymerase activity. Molecular identity of the three activities is further indicated by their comparative rates of thermal inactivation and their sensitivity to ionic strength. The 3-5 exonuclease activity hydrolyzes only single-standed DNA. The rate of hydrolysis is twice the optimal rate of polymerization. The products are 5-mononucleotides, but the 3-5 activity is unable to cleave free dinucleotides or the 5-terminal dinucleotide of a polydeoxynucleotide chain. The 3-5 activity will not degrade 3-phosphoryl-terminated oligonucleotides such as d(pTpTpTp). The 5-3 activity catalyzes the hydrolysis of single-stranded DNA at 1/15 the rate of the 3-5 exonuclease. The 5-3 exonuclease requires the presence of a 5 single-stranded terminus in order to initiate hydrolysis, but will thereafter proceed into a double-stranded region. Although the limit products found during hydrolysis of substrates designed to assay specifically the 5-3 activity are predominantly mono- and dinucleotides, these products probably arise from the subsequent hydrolysis of oligonucleotides by the 3-5 hydrolytic activity. This interpretation is supported by (a) the relatively greater activity of the 3-5 exonuclease, (b) the inability of the enzyme to degrade d(pTpTpTp), and (c) the release of the 5 terminus of a single-stranded DNA molecule as an oligonucleotide. The 5-3 exonuclease attacks ultraviolet-irradiated duplex DNA which has first been incised by the Micrococcus luteus endonuclease specific for thymine dimers in DNA.     

Parallel stranded duplex DNA.

Three linear 21-nt oligonucleotides (C2, C3, C7) have been synthesized with different sequences of A and T residues. One pairwise combination, (C3, C7), hybridizes to form a conventional antiparallel duplex (aps-C3.C7), whereas the pair C2, C3 forms a duplex (ps-C2.C3) in which the two strands are in a parallel orientation and the A.T base-pairs in a reverse Watson-Crick configuration. The existence of the novel ps helical structure was established from the following criteria: (i) The electrophoretic mobilities of the ps and aps duplexes in native and denaturing polyacrylamide gels are similar. (ii) The ps duplex is not a substrate for T4 DNA ligase. (iii) Salt-dependent thermal transitions are observed for the two duplexes, but the melting temperatures of the ps molecules are 15 degrees C lower. (iv) The ultraviolet absorption and circular dichroism spectra of the ps duplex are indicative of a base-paired structure, but differ systematically from that of the aps helix. (v) Based on fluorescent measurements, the bis-benzimidazole drug BBI-258 shows a lower affinity for the ps compared to the aps duplex, whereas the opposite preference holds for the intercalator ethidium bromide. We conclude from the present study that parallel stranded DNA is a stable conformation which can arise by interaction between two conventional strands with appropriate sequence homology.     

Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.     

An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme

Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.     

DNA polymerase I and a protein complex bind specifically to E. coli palindromic unit highly repetitive DNA: implications for bacterial chromosome organization.

Starting from a crude E. coli extract, two activities which specifically protect highly repetitive bacterial DNA sequences (called PU for Palindromic Unit or REP for Repetitive Extragenic Palindromic sequence) against a digestion with Exonuclease III have been purified. We show that one of these activities is due to the DNA polymerase I (Pol I). This constitutes the first indication for a specific interaction between Pol I and a duplex DNA. This interaction requires the presence of PU. It was confirmed and analyzed by native gel electrophoresis and DNase I footprinting experiments. The other activity contained at least five polypeptides. Its binding to PU DNA sequences was confirmed by native gel electrophoresis. Implications for the possible origin and functions of PU are discussed.