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1.
Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions. 相似文献
2.
PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase 总被引:2,自引:0,他引:2
The tRNALys anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129–143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNALys recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised. 相似文献
3.
The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini. Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition. We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC. The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease. Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations. Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase. 相似文献
4.
Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.
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Anticodon nuclease is a bacterial restriction enzyme directed against tRNA(Lys). We report that anticodon nuclease also cleaves mammalian tRNA(Lys) molecules, with preference and site specificity shown towards the natural substrate. Expression of the anticodon nuclease core polypeptide PrrC in HeLa cells from a recombinant vaccinia virus elicited cleavage of intracellular tRNA(Lys),3. The data justify an inquiry into the possible application of anticodon nuclease as an inhibitor of tRNA(Lys),3-primed HIV replication. They also indicate that the anticodon region of tRNA(Lys) is a substrate recognition site and suggest that PrrC harbors the enzymatic activity. 相似文献
5.
Amitsur M Benjamin S Rosner R Chapman-Shimshoni D Meidler R Blanga S Kaufmann G 《Molecular microbiology》2003,50(1):129-143
The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection. 相似文献
6.
The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4‐excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction‐modification (RM) protein and turned on by a phage‐encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double‐stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild‐type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction. 相似文献
7.
Klipcan L Moor N Finarov I Kessler N Sukhanova M Safro MG 《Journal of molecular biology》2012,415(3):527-537
Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 Å. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1–C72 base pair, and the “discriminator” base A73, proposed to contribute to tRNAPhe identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS–tRNAPhe provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the “closed” to the productive “open” state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions. 相似文献
8.
Preyesh Stephen Sheng Ye Ming Zhou Jian Song Rongguang Zhang En-Duo Wang Richard Giegé Sheng-Xiang Lin 《Journal of molecular biology》2018,430(11):1590-1606
Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. 相似文献
9.
The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3' OH terminus 总被引:15,自引:4,他引:11
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The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL3, and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron. The second, more compact domain, is composed of the five other arms and most probably contains the structure recognized by valyl-tRNA synthetase. In this domain three successive arms strikingly resemble the T[unk], anticodon, and D arms found in tRNA. Near the amino-acid accepting terminus, however, there is a new stem and loop region not found in standard tRNA. This secondary structure is compatible with a L-shaped three-dimensional organization in which the corner of the L and the anticodon-containing limb are similar to, and the amino-acid accepting region different from, that in tRNA. Ethylnitrosourea accessibility studies have shown similar tertiary structure features in the T[unk] loop of tRNAVal and in the homologous region of the viral RNA. 相似文献
10.
Klaiman D Amitsur M Blanga-Kanfi S Chai M Davis DR Kaufmann G 《Nucleic acids research》2007,35(14):4704-4714
The optional Escherichia coli restriction tRNase PrrC represents a family of potential antiviral devices widespread among bacteria. PrrC comprises a functional C-domain of unknown structure and regulatory ABC/ATPase-like N-domain. The possible involvement of a C-domain sequence in tRNALys recognition was investigated using a matching end-protected 11-meric peptide. This mimic, termed here LARP (Lys-anticodon recognizing peptide) UV-cross-linked tRNALys anticodon stem-loop (ASL) analogs and inhibited their PrrC-catalyzed cleavage. Trimming LARP or introducing in it inactivating PrrC missense mutations impaired these activities. LARP appeared to mimic its matching protein sequence in ability to dimerize in parallel, as inferred from the following results. First, tethering Cys to the amino- or carboxy-end of LARP dramatically enhanced the ASL-cross-linking and PrrC-inhibiting activities under suitable redox conditions. Second, Cys-substitutions in a C-domain region containing the sequence corresponding to LARP elicited specific intersubunit cross-links. The parallel dimerization of PrrC's C-domains and expected head-to-tail dimerization of its N-domains further suggest that the NTPase and tRNALys-binding sites of PrrC arise during distinct assembly stages of its dimer of dimers form. 相似文献
11.
Lambda bacteriophage containing yeast tyrosine transfer RNA genes were prepared by molecular recombination. These phage were identified by hybridization of 125I-labeled yeast tRNATyr to plaques from lambda-yeast recombinant phage pools. The cloned yeast EcoRI fragments that hybridize to 125I-labeled tRNATyr were compared in size with the fragments in total yeast DNA that hybridize to the same probe. These comparisons indicate that seven of the eight different tRNATyr genes have been isolated. Unambiguous evidence that these seven fragments contain tRNATyr coding regions was obtained by showing that they hybridize to aminoacylated [3H]Tyr-tRNATyr. Only one of the fragments hybridizes to 32P-labeled total yeast tRNA in the presence of competing unlabeled tRNATyr; the tRNATyr genes, therefore, are not predominantly organized into heteroclusters of tRNA genes. 相似文献
12.
Mom Das Oscar Vargas-Rodriguez Yuki Goto Hiroaki Suga Karin Musier-Forsyth 《Nucleic acids research》2014,42(6):3943-3953
Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNAPro have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNAPro in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNAPro. Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells. 相似文献
13.
The conserved bacterial protein RloC, a distant homologue of the tRNALys anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn++ -responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans . As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage. 相似文献
14.
Yulan Xiong Candice E. Coombes Austin Kilaru Xiaojie Li Aaron D. Gitler William J. Bowers Valina L. Dawson Ted M. Dawson Darren J. Moore 《PLoS genetics》2010,6(4)
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson''s disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker''s yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration. 相似文献
15.
Yue Jiang Shani Blanga Michal Amitsur Roberto Meidler Eli Krivosheyev Mallikarjun Sundaram Ashok C Bajji Darrell R Davis Gabriel Kaufmann 《The Journal of biological chemistry》2002,277(6):3836-3841
The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t(6)A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) has a weaker positive effect depending on the context of other modifications. The s(2)U34 modification apparently has none and the pseudouridine (psi39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp(287) contacts mnm(5)s(2)U34 was reinforced by the observations that the mammalian tRNA(Lys-3) wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm(5)s(2)U) is inhibitory and that the D287H mutant favors tRNA(Lys-3) over Escherichia coli tRNA(Lys). The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA(Lys-3) primer molecules annealed to the genomic RNA template of the human immunodeficiency virus. 相似文献
16.
Oscar Vargas-Rodriguez Marina Bakhtina Daniel McGowan Jawad Abid Yuki Goto Hiroaki Suga Karin Musier-Forsyth 《The Journal of biological chemistry》2020,295(48):16180
Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNAPro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate. 相似文献
17.
Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)+ RNA export and in vivo poly (A)+ RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains. 相似文献
18.
Klaiman D Steinfels-Kohn E Krutkina E Davidov E Kaufmann G 《Nucleic acids research》2012,40(17):8568-8578
The conserved bacterial anticodon nuclease (ACNase) RloC and its phage-excluding homolog PrrC comprise respective ABC-adenosine triphosphatase (ATPase) and ACNase N- and C-domains but differ in three key attributes. First, prrC is always linked to an ACNase silencing, DNA restriction-modification (R-M) locus while rloC rarely features such linkage. Second, RloC excises its substrate's wobble nucleotide, a lesion expected to impede damage reversal by phage transfer RNA (tRNA) repair enzymes that counteract the nick inflicted by PrrC. Third, a distinct coiled-coil/zinc-hook (CC/ZH) insert likens RloC's N-region to the universal DNA damage checkpoint/repair protein Rad50. Previous work revealed that ZH mutations activate RloC's ACNase. Data shown here suggest that RloC has an internal ACNase silencing/activating switch comprising its ZH and DNA-break-responsive ATPase. The existence of this control may explain the lateral transfer of rloC without an external silencer and supports the proposed role of RloC as an antiviral contingency acting when DNA restriction is alleviated under genotoxic stress. We also discuss RloC's possible evolution from a PrrC-like ancestor. 相似文献
19.
Two novel phenotypes previously associated with arl mutations of Escherichia coli, increased frequencies of genetic recombination and unusual sensitivity of DNA to the single-strand-specific nuclease S1, have been defined most completely by the properties of λ bacteriophages grown on arl bacteria (Arl? phages). We now find that plasmids maintained in arl mutants (Arl? plasmids) exhibit elevated recombination frequencies, unusual sensitivity to nuclease S1 (in a limited number of regions) and a new Arl phenotype, partially deficient methylation of the inner cytosine at C-C-(A/T)-G-G sequences.A variety of Arl? plasmids (all pBR322 derivatives) show elevated recombination (4 to 10-fold) by three different assays (frequencies of homomultimers and of heteromultimers, efficiency of intramolecular recombination). Plasmids from arl bacteria (after conversion to linear form) are nicked by nuclease S1 about 0.7 times per duplex; Arl+ plasmids are nuclease S1-resistant. Restriction endonuclease EcoRII (recognition sequence, C-C-(A/T)-G-G) cuts Arl? plasmid DNA more readily than Arl+ DNA, but Arl? plasmids are still more EcoRII-resistant than Dcm? plasmids (from E. coli dcm mutants, which lack the chromosomal cytosine methylase; recognition sequence, also C-C-(A/T)-G-G). By chromatographic analyses, Arl? plasmid DNA contains less 5-methylcytosine than Arl+ (0.07% versus 0.15%). although the 6-methyladenine content is the same (0.5mol%). 相似文献
20.
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. 相似文献