Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC

tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in a eukaryon in the absence of other bacterial or viral proteins. Whereas Streptococcus PrrC is also toxic in yeast, Neisseria and Xanthomonas PrrCs are not. Via analysis of the effects of 118 mutations on EcoPrrC toxicity in yeast, we identified 22 essential residues in the NTPase domain and 11 in the nuclease domain. Overexpressing PrrCs with mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC. Our findings support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the NTPase domains to form two composite NTP phosphohydrolase sites. Comparisons of EcoPrrC activity in a variety of yeast genetic backgrounds, and the rescuing effects of tRNA overexpression, implicate tRNA^Lys(UUU) as a target of EcoPrrC toxicity in yeast.     

Restriction insensitivity in bacteriophage T5. III. Characterization of EcoRI-sensitive mutants by restriction analysis.

Despite the fact that its DNA carries six EcoRI cleavage sites, bacteriophage T5 is able to grow on an EcoRI restricting host, suggesting that it specifies a restriction protection system. In the hope of identifying this protection system, mutants of T5 have been isolated which are unable to grow on an EcoRI restricting host. Analysis of the DNA of such mutants shows that they have each acquired two new EcoRI sites per molecule as a consequence of a single EcoRI site (ris) mutation located in the terminally repetitious, first step transfer (FST) region of the genome. The EcoRI sites generated by the ris mutations differ from the natural EcoRI sites in that the latter are situated on the second step transfer (SST) DNA, which suggests that the in vivo sensitivity of ris mutants is a consequence of having an EcoRI site on the FST DNA. This is understandable, if the hypothetical restriction protection genes are also located on the FST DNA. While expression of these genes would protect natural sites on the SST DNA, the ris sites would, on the contrary, enter an environment in which the protection, products had not yet been synthesized.Construction of double and triple ris mutants has allowed the ordering of the ris sites and the construction of an EcoRI restriction map of the FST region. In addition, the ris mutants allow estimation of the size of the terminal repetition of T5 DNA as 5.9 × 10⁶ to 6.0 × 10⁶ daltons. Correlation of the physical map of the FST region with the already established genetic map of this region allows orientation of the pre-early genes on the genetic and physical maps, and approximate localization of two amber mutations on the physical map.     

Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr₁ to hr₅, occur at or near the following EcoRI fragment junctions: hr₁EcoRI-B—EcoRI-I (0.0 map units); hr₂, EcoRI-A—EcoRI-J (19.8 map units); hr₃, EcoRI-C—EcoRI-G (52.9 map units); hr₄, EcoRI-Q—EcoRI-L (69.8 map units); and hr₅, EcoRI-S—EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr₂ and hr₅, respectively. The sizes of the homologous regions were about 800 base pairs for hr₂, 500 base pairs for hr₅, and less than 500 base pairs for hr₁, hr₃, and hr₄. A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.     

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI¹, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI¹ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI¹ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI¹ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.     

The DNA sequence on bacteriophage G4 recognized by the Escherichia coli B restriction enzyme.

Bacteriophage G4 possesses a single EcoB site located in the overlap between restriction fragments HinfI-12 and HaeIII-6. The sequence 5′-T-G-A … 8N … T-G-C-T occurs once in this segment and nowhere else in the DNA sequence of G4. Four independent G4 mutants that were not restricted by Escherichia coli B possessed the sequence 5′-T-G-A … 8N … T-G-C-C. The common sequence shared by the previously mapped EcoB sites on φXsB1, simian virus 40, f1, and fd DNAs is 5′-T-G-A … 8N … T-G-C-T … 9N … T. However, the sequence in the region of the G4 EcoB site contains an A instead of the final T conserved in these other examples. When the G4 EcoB site is aligned with the other EcoB sites, there are no conserved residues within 50 bases of the common sequence, 5′-T-G-A … 8N … T-G-C-T, except for those seven residues. The analysis of the EcoB site on G4 provides further evidence that only those seven bases are recognized by the E. coli B restriction enzyme.     

Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.     

EcoRI analysis of bacteriophage P22 DNA packaging.

Bacteriophage P22 linear DNA molecules are a set of circularly permuted sequences with ends located in a limited region of the physical map. This mature form of the viral chromosome is cut in headful lengths from a concatemeric precursor during DNA encapsulation. Packaging of P22 DNA begins at a specific site, which we have termed pac, and then proceeds sequentially to cut lengths of DNA slightly longer than one complete set of P22 genes (Tye et al., 1974b). The sites of DNA maturation events have been located on the physical map of EcoRI cleavage sites in P22 DNA. EcoRI digestion products of mature P22 wild-type DNA were compared with EcoRI fragments of two deletion and two insertion mutant DNAs. These mutations decrease or increase the length of the genome, but do not alter the DNA encapsulation mechanism. Thus the position of mature molecular ends relative to EcoRI restriction sites is different in each mutant, and comparison of the digests shows which fragments come from the ends of linear molecules. From the positions of the ends of molecules processed in sequential headfuls, the location of pac and the direction of encapsulation relative to the P22 map were deduced. The pac site lies in EcoRI fragment A, 4.1 × 10³ base-pairs from EcoRI cleavage site 1. Sequential packaging of the concatemer is initiated at pac and proceeds in the counterclockwise direction relative to the circular map of P22. One-third of the linears in a population are cut from the concatemer at pac, and most packaging sequences do not extend beyond four headfuls.Fragment D is produced by EcoRI cleavage at a site near the end of a linear chromosome which has been encapsulated starting at pac. The position of the pac site is therefore defined by one end of fragment D. The pac site is not located near genes 12 and 18, the only known site for initiation of P22 DNA replication, but lies among late genes at a position on the physical gene map approximately analogous to the cohesive end site (cos) of bacteriophage λ at which λ DNA is cleaved during encapsulation. Our results suggest that P22 and λ DNA maturation mechanisms have many common properties.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Functional cooperation between exonucleases and endonucleases--basis for the evolution of restriction enzymes

Many types of restriction enzymes cleave DNA away from their recognition site. Using the type III restriction enzyme, EcoP15I, which cleaves DNA 25–27 bp away from its recognition site, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only a single round of DNA cleavage. Significantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage. This observation may hold true for all restriction enzymes cleaving DNA sufficiently far away from their recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. Based on results presented here and other data on well-characterised restriction enzymes, a functional evolutionary hierarchy of restriction enzymes is discussed.     

C.EcoO109I,a regulatory protein for production of EcoO109I restriction endonuclease,specifically binds to and bends DNA upstream of its translational start site

The EcoO109I restriction-modification system, which recognizes 5′-(A/G)GGNCC(C/T)-3′, has been cloned, and contains convergently transcribed endonuclease and methylase. The role and action mechanism of the gene product, C.EcoO109I, of a small open reading frame located upstream of ecoO109IR were investigated in vivo and in vitro. The results of deletion analysis suggested that C.EcoO109I acts as a positive regulator of ecoO109IR expression but has little effect on ecoO109IM expression. Assaying of promoter activity showed that the expression of ecoO109IC was regulated by its own gene product, C.EcoO109I. C.EcoO109I was overproduced as a His-tag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity. C.EcoO109I exists as a homodimer, and recognizes and binds to the DNA sequence 5′-CTAAG(N)₅CTTAG-3′ upstream of the ecoO109IC translational start site. It was also shown that C.EcoO109I bent the target DNA by 54 ± 4°.     

小叶锦鸡儿基因组DNA的提取及AFLP反应体系的建立

以小叶锦鸡儿幼嫩叶片为材料,采用改良的SDS法提取其基因组DNA,通过优化AFLP技术体系中的几个主要因素,建立了适合小叶锦鸡儿的AFLP银染反应体系。改良的SDS法能通过在提取液中加入β-巯基乙醇防止氧化、加入PVP去除酚类等物质,获得满足AFLP分析要求的纯度高、完整性好的基因组DNA;用EcoRⅠ和MseⅠ37 ℃双酶切4 h后可以将500 ng的基因组DNA完全切开。酶切产物和接头经16℃连接过夜后,用带有1个选择性碱基的引物和带有3个选择性碱基的引物分别进行预扩增和选择性扩增,扩增产物经变性聚丙烯酰胺凝胶电泳分离,用AgNO₃染色,得到了清晰的指纹式样。小叶锦鸡儿AFLP反应体系的建立为利用该技术研究小叶锦鸡儿的遗传多样性奠定了实验基础。     

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30°C and 42°C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 Å resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.     

Probing of contacts between <Emphasis Type="Italic">Eco</Emphasis>RII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.     

Complete Sequence and Enhancer Function of the Homologous DNA Regions of Autographa californica Nuclear Polyhedrosis Virus

The nucleotide sequence of the five regions of homologous DNA in the genome of Autographa californica nuclear polyhedrosis virus DNA was determined. The homology of repeated sequences within a region was 65 to 87%, and the consensus sequences for each region were 88% homologous to each other. Sequences proximal to the EcoRI sites were most conserved, while the distal sequences were least conserved. The EcoRI sites formed the core of a 28-base-pair imperfect inverted repeat. All homologous regions functioned as enhancers in a transient expression assay. A single EcoRI minifragment located between EcoRI-Q and -L enhanced the expression of 39CAT as efficiently as the regions containing numerous EcoRI repeats did.     

FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease

We found unusual reactivity of a heterochiral dodecadeoxynucleotide toward EcoRI that contains an unnatural l-nucleotide residue at the 3′-flanking site of the hexameric EcoRI recognition sequence. In this study, the kinetic parameters for the reaction were determined by means of a heterochiral molecular beacon that contains the EcoRI recognition sequence in the stem region and possesses the fluorescent and quenching dyes at the 5′- and 3′-termini, respectively. We found that the heterochiral molecular beacon showed large V_max and small K_m values compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.