Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

The Sse9I restriction-modification system: organization of genes and comparative analysis of proteins structures

A nucleotide sequence was established for the full-length Sporosarcina species 9D operon coding for enzymes of type II restriction-modification system Sse9I. These enzymes recognize the tetranucleotide DNA sequence 5'-AATT-3'. The operon was shown to consist of three genes that are situated with the order: sse9IC-sse9IR-sse9IM and are transcribed in the same direction. These genes encode the control protein (C.Sse9I), restriction endonuclease (R.Sse9I) and DNA-methyltransferase (M.Sse9I), respectively. A specific DNA sequence (C-box) presumably recognized by C-protein was found immediately upstream of sse9IC gene. The comparative analysis of amino acid sequences of C.Sse9I and R.Sse9I with those of relative proteins has been done. It was found that R.Sse9I revealed the most homology with the segments of R.MunI (5'-CAATTG-3') and R.EcoRI (5'-GAATTC-3'), where amino acid residues, responsible for recogniton of AATT core sequence are located. The sse9IR gene was cloned into the temperature-inducible expression vector, and recombinant Sse9I restriction endonuclease preparation was isolated.     

Restriction enzyme BstZ17I is sensitive to cytosine methylation

The cleavage patterns of a subset of restriction enzymes are blocked or impaired when a methylated CpG is overlapped with either the 5' or 3' end of the canonical restriction site. BstZ17I restriction endonuclease is a blunt-end cutter, which recognises the hexanucleotide sequence GTA(downward arrow)TAC. In this report, I show that the BstZ17I restriction enzyme is sensitive to cytosine methylation. Using both in vitro-methylated episomal plasmids and lambdaDNA, I demonstrate that the BstZ17I restriction enzyme is sensitive to cytosine methylation that occurs 3' and/or 5' of the canonical recognition sequence.     

Physical map of the Listeria monocytogenes chromosome.

The circular physical map of the pathogenic bacterium Listeria monocytogenes LO28 (serovar 1/2c) was established by using pulsed-field gel electrophoresis. The L. monocytogenes chromosome contains eight NotI fragments of 1,100, 940, 400, 335, 280, 45, 30, and 20 kb in size and eight Sse8387I fragments of 860, 680, 680, 370, 335, 130, 70, and 25 kb. Therefore, the total length of the genome is 3,150 kb. To order the NotI fragments on the chromosome, we used a strategy which can be of general use. We first cloned chromosomal HindIII or EcoRI fragments in pBR322. DNA extracted from the total libraries was digested by NotI and ligated to a NotI-kanamycin resistance cassette obtained by cutting Tn5 with NotI. After transformation in Escherichia coli, kanamycin-resistant clones originating from NotI-containing EcoRI or HindIII fragments were isolated. The two EcoRI-NotI or HindIII-NotI fragments of each recombinant plasmid were isolated and used as probes on Southern blot hybridizations to identify and link the corresponding NotI fragments. Seven NotI fragments were ordered in this way. The last junction was demonstrated by partial digest analysis. All L. monocytogenes genes identified so far as well as the six rRNA operons were localized on the NotI map. Regions homologous to genes from closely related bacteria were also detected and localized. Southern blot analysis of simple Sse8387I digests or double Sse8387I-NotI digests probed with the various NotI probes allowed us to align the Sse8387I fragments and localize the single SfiI site, resulting in the establishment of the first genetic and physical map of the L. monocytogenes chromosome.     

Cleavage of a model DNA replication fork by a Type I restriction endonuclease

Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.     

Construction of physical and genetic maps of Chlamydia trachomatis serovar L2 by pulsed-field gel electrophoresis.

We constructed the physical map of Chlamydia trachomatis serovar L2 by using three restriction endonucleases, NotI (GC[GGCCGC), SgrAI (C(A/G)[CCGG(T/G)G), and Sse8387I (CCTGCA[GG), and we analyzed the fragments by pulsed-field gel electrophoresis. A total of 25 restriction endonuclease sites and 13 genes and/or operons were located on the map. The genome size was determined to be 1,045 kb. Neither highly transcribed chlamydia genes nor developmental cycle-specific genes were clustered on the genome.     

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Distribution of CWG and CCWGG in the Human Genome

Expression of the bacterial CG methyltransferase M?HhaI in mammalian cells appears to generate significant biological effects, while biological effects of the expression of the non-CG methyltransferase M?EcoRII in human cells have not been detected. The association of cytosine methylation with the CG site in mammals is also associated with clustering of CG sites near 5´ control regions (CG-islands) of human genes. Moreover spontaneous deamination of 5-methylcytosine at these sites is thought to lead to the well known deficiency of CG sites in genomes where endogenous CG methyltransferases are expressed. Since these associations are generally taken to imply a biological function for the CG dinucleotide that is associated with its selective methylation by endogenous DNA methylation systems, we have asked whether or not CWG or CCWGG sites are clustered in regions flanking human genes and whether or not an overall deficiency of CWG or CCWGG occurs in the human genome. Using build 36.1, of the human genome, we inspected the regions flanking the 28,501 well known gene loci in the human genome. Our analysis confirmed the expected clustering of CG sites near the 5´ region of known genes and open reading frames. In contrast to the CG site, neither the CWG site nor the CCWGG site recognized by the bacterial methyltransferase M?EcoRII were clustered in any particular region near known genes and open reading frames. Moreover, neither the CCWGG nor the CWG site was depleted in the human genome, again in sharp contrast to the known genomic deficiency of CpG sites. Our findings suggest that in contrast to CG site recognition, human cytosine methyltransferases recognize CWG and CCWGG only at very low frequency if at all.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Purification of Mbo II methylase (GAAGmA) from Moraxella bovis: site specific cleavage of DNA at nine and ten base pair sequences.

The restriction modification methylase M. Mbo II has been purified using a sensitive oligonucleotide linker assay. The enzyme methylates the Mbo II recognition sequence* GAAGA at adenine to produce GAAGmA. M. Mbo II can be used in conjunction with the methylation dependent restriction endonuclease Dpn I (GmATC) to produce cleavage at the 10 base sequence GAAGATCTTC. When M. Mbo II is used in combination with M. Cla I (ATCGATCGAT), cleavage by Dpn I occurs at the four ten base sequences GAAGATCTTC, GAAGATCGAT, ATCGATCTTC and ATCGATCGAT, which is equivalent to a nine base recognition site. The use of combinations of adenine methylases and Dpn I to generate highly selective DNA cleavages at a variety of sequences up to fourteen base pairs is discussed.     

Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT)

Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (～160 kDa), responsible for methylation of DNA, and the restriction endonuclease (～400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT), based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7)TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NT)in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.     

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.     

Mutational analysis of two putative catalytic motifs of the type IV restriction endonuclease Eco57I

The role of two sequence motifs (SM) as putative cleavage catalytic centers (77)PDX(13)EAK (SM I) and (811)PDX(20)DQK (SM II) of type IV restriction endonuclease Eco57I was studied by site-directed mutational analysis. Substitutions within SM I; D78N, D78A, D78K, and E92Q reduced cleavage activity of Eco57I to a level undetectable both in vivo and in vitro. Residual endonucleolytic activity of the E92Q mutant was detected only when the Mg(2+) in the standard reaction mixture was replaced with Mn(2+). The mutants D78N and E92Q retained the ability to interact with DNA specifically. The mutants also retained DNA methylation activity of Eco57I. The properties of the SM I mutants indicate that Asp(78) and Glu(92) residues are essential for cleavage activity of the Eco57I, suggesting that the sequence motif (77)PDX(13)EAK represents the cleavage active site of this endonuclease. Eco57I mutants containing single amino acid substitutions within SM II (D812A, D833N, D833A) revealed only a small or moderate decrease of cleavage activity as compared with wild-type Eco57I, indicating that the SM II motif does not represent the catalytic center of Eco57I. The results, taken together, allow us to conclude that the Eco57I restriction endonuclease has one catalytic center for cleavage of DNA.     

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.     

The inhibition of restriction endonuclease PvuII cleavage activity by methylation outside its recognition sequence.

The recombinant plasmid pGEM4Z-ras DNA which was methylated on dam and dcm sites outside the PvuII recognition sequence was digested with restriction endonuclease PvuII, and one of the three PvuII sites was about 16-fold less efficient to cleave than either of the other two. On the contrary, the three PvuII sites were cleaved at about the same rate on the unmethylated DNA molecule. The results show that the cleavage inhibition of the methylated DNA on the certain PvuII site was caused by methylation outside the PvuII recognition sequence. Maybe a adjacent methylated dam site *A was responsible for the less efficient cleavage. This observation suggests that methylation outside the recognition sequence may be considered a new factor in the kinetic experiment of restriction endonuclease.     

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.     

Characterisation of the novel restriction endonuclease <Emphasis Type="Italic">Sui</Emphasis>I from <Emphasis Type="Italic">Sulfolobus islandicus</Emphasis>

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70°C.     

Reactions of type II restriction endonucleases with 8-base pair recognition sites

Type II restriction endonucleases usually recognize 4-6-base pair (bp) sites on DNA and cleave each site in a separate reaction. A few type II endonucleases have 8-bp recognition sites, but these seem unsuited for restriction, since their sites are rare on most DNA. Moreover, only one endonuclease that recognizes a target containing 8 bp has been examined to date, and this enzyme, SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were tested on plasmids that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and SdaI all acted through equal and independent reactions at each site. AscI cleaved the DNA with one site at the same rate as that with two sites but acted processively on the latter. In contrast, SgrAI showed a marked preference for the plasmid with two sites and cleaved both sites on this DNA in a concerted manner, like SfiI. Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate requirement, they may function in DNA restriction.     

Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methylcytosine with MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases.

The cleavage specificity of R.Cfr9I was determined to be C decreases CCGGG whereas the methylation specificity of M.Cfr9I was C4mCCGGG. The action of MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligonucleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation, in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonucleases. The cleavage of methylated substrates with restriction endonucleases is discussed.