DNA-dependent Reaction Research Articles

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space: IV. VIRAL PROTEINASE SLIDES ALONG DNA TO LOCATE AND PROCESS ITS SUBSTRATES

Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 10(6) bp(2)/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space: II. ADENOVIRUS PROTEINASE IS ACTIVATED IN AN UNUSUAL ONE-DIMENSIONAL BIOCHEMICAL REACTION

Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 × 10(6) bp(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.

Chromatin dynamics mediated by histone modifiers and histone chaperones in postreplicative recombination

Eukaryotic chromatin is regulated by chromatin factors such as histone modification enzymes, chromatin remodeling complexes and histone chaperones in a variety of DNA-dependent reactions. Among these reactions, transcription in the chromatin context is well studied. On the other hand, how other DNA-dependent reactions, including postreplicative homologous recombination, are regulated in the chromatin context remains elusive. Here, histone H3 Lys56 acetylation, mediated by the histone acetyltransferase Rtt109 and the histone chaperone Cia1/Asf1, is shown to be required for postreplicative sister chromatid recombination. This recombination did not occur in the cia1/asf1-V94R mutant, which lacks histone binding and histone chaperone activities and which cannot promote the histone acetyltransferase activity of Rtt109. A defect in another histone chaperone, CAF-1, led to an increase in acetylated H3-K56 (H3-K56-Ac)-dependent postreplicative recombination. Some DNA lesions recognized by the putative ubiquitin ligase complex Rtt101-Mms1-Mms22, which is reported to act downstream of the H3-K56-Ac signaling pathway, seem to be increased in CAF-1 defective cells. Taken together, these data provide the framework for a postreplicative recombination mechanism controlled by histone modifiers and histone chaperones in multiple ways.

Werner Syndrome Protein Contains Three Structure-specific DNA Binding Domains

Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3'-5'-exonuclease, an ATPase, and a 3'-5'-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5'-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one N-terminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways.

Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of nucleotide binding site mutant.

Gene 4 of bacteriophage T7 encodes two co-linear proteins, the 56- and 63-kDa gene 4 proteins. The 56-kDa protein translocates 5' to 3' on single-stranded DNA using nucleotide hydrolysis for energy and is a helicase. The 63-kDa gene 4 protein catalyzes all of the activities of the 56-kDa gene 4 protein and, in addition, catalyzes the synthesis of oligoribonucleotides on single-stranded DNA. Two conserved residues in a putative nucleotide binding site of the 63-kDa gene 4 protein were mutated by substituting Val and Met for wild-type residues Gly and Lys, at positions 317 and 318, respectively. The mutant 63-kDa gene 4 protein lacks the ability to catalyze the hydrolysis of a nucleoside 5'-triphosphate in a single-stranded DNA-dependent reaction and inhibits nucleotide hydrolysis by wild-type gene 4 proteins. The mutant primase contains 0.4% of the primase activity of the 63-kDa gene 4 protein on M13 single-stranded DNA and 12% of the wild-type primase activity on an oligonucleotide with a single primase recognition site. Addition of wild-type 56-kDa gene 4 protein stimulates the mutant primase activity over 50-fold on M13 single-stranded DNA and 8-fold on oligonucleotides. This increase in primase activity correlates with an increase in the affinity of the mutant primase-wild-type helicase complex for single-stranded DNA template.

A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin.

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

The Assembly of Regularly Spaced Nucleosomes in the Xenopus Oocyte S-150 Extract Is Accompanied by Deacetylation of Histone H4

Histone proteins, which were assembled into chromatin using the Xenopus oocyte S-150 extract, were analyzed on acid-urea gels and Triton-acid-urea gels to determine their state of modification. We find that histone H4, which is present in a diacetylated form in the oocyte S-150, gradually loses its acetate groups as the DNA is packaged into chromatin. Thus, this process parallels the one observed in vivo during chromatin formation in growing eucaryotic cells. Histone H4 deacetylation in the oocyte S-150 is a DNA-dependent reaction. This reaction is blocked when butyrate (an inhibitor of histone deacetylase) is added at the onset of the chromatin assembly process. When butyrate is added at the end of the assembly process, no de novo acetylation of the nucleosomal histone H4 is observed. Chromatin with regularly spaced nucleosomes, displaying periodicities ranging from 160 to 220 base pairs, can be assembled in vitro with the oocyte S-150 (Rodríguez-Campos, A., Shimamura, A., and Worcel, A. (1989) J. Mol. Biol., in press). This chromatin may contain either deacetylated histone H4 when assembled under standard conditions or diacetylated H4 when assembled in the presence of butyrate. Both types of chromatin display identical structures upon digestion with nucleases. The potential applications of this system toward the study of the naturally occurring diacetylated histone H4 are discussed.

Intrinsic DNA-dependent ATPase activity of reverse gyrase.

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Hydrolysis of UTP.

Hydrolysis of UTP catalyzed by the recA protein of Escherichia coli is stimulated by both double- (DS) and single-stranded (SS) DNA. DS DNA-dependent UTPase activity has a sharp optimum near pH 6. SS DNA-dependent UTP hydrolysis also is optimal near pH 6, although considerable activity remains at pH 8. Both SS and DS DNA-dependent UTPase activities are nonlinearly dependent on recA protein concentration at pH 6 but the SS DNA-dependent reaction shows a linear dependence on enzyme concentration at pH 8. The Km for UTP in the SS DNA-dependent reaction decreases from 147 microM at pH 8 to 33 microM at pH 6. The Km for UTP in the DS DNA-dependent reaction is 247 microM at pH 6. In addition, the Hill coefficient for UTP in the SS DNA-dependent reaction decreases from 3.5 at pH 8 to 1.9 at pH 6, while in the DS DNA-dependent reaction, the Hill coefficient is 2.4 at pH 6. Thus, the UTPase activity of the recA protein differs from the ATPase activity of recA protein primarily in the pH dependence of KmUTP, Vmax, and response to enzyme concentration of the SS DNA-dependent hydrolysis reaction. These differences may be related to the inability of UTP to substitute effectively for ATP in recA protein-promoted annealing and assimilation of SS DNA.

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis.

Both single- and double-stranded DNA stimulate the hydrolysis of ATP catalyzed by the recA protein of Escherichia coli. However, the reactions differ in their pH optima, response to recA protein concentration, salt sensitivity, and degree of inhibition by ADP, all of which reflect different requirements for the prehydrolytic binding of single- and double-stranded DNA by the RecA protein. Single- and double-stranded DNA stimulate hydrolysis of the same nucleoside triphosphates, principally (r,d)ATP and (r,d)UTP, suggesting that a single hydrolytic site is utilized in both single- and double-stranded DNA-dependent reactions. recA protein also catalyzes detectable ATP hydrolysis in the absence of exogenous DNA, although the rate is reduced 2 to 3 orders of magnitude. This DNA-independent hydrolysis shows the same nucleotide specificity at pH 6.2 and 7.5, although the rate of hydrolysis depends upon the pH.

Novel Properties of DNA Polymerase β with Poly(rA). oligo(dT) Template-Primer1

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.

A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments

Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E. coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA results from a DNA-dependent reaction.

Effects of polyamines on in vitro dna synthesis by DNA polymerases from calf thymus.

DNA synthesizing reactions catalyzed both large and small species of calf thymus DNA polymerase (DNA polymerase-alpha and -beta) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0 mM for spermidine and 2-10 mjM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows: 1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used. 2) The nautral DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated spermidine even at the optimal concentration of Mn2+. 3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KC1 or NaC1 at concentrations less than 1/100 of those of salts.

Conversion of phichi174 and fd DNA to their replicative forms by two enzyme systems in Escherichia coli.

Extracts of Escherichia coli pol Al strains catalyze the conversion of φX174 or fd (M13) single-stranded circular DNA to the replicative form (1,2,). The fd DNA dependent reaction is sensitive to rifampicin and requires all four ribo- nucleoside triphosphates, but does not involve the products of the dnaA or dnaB gene. The reaction with φX174 DNA, in contrast, is rifampicin resistant and is temperature sensitive in extracts of dnaBts cells (1,2).

Conversion of phiX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli.

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.