Evolutionary relationship of<Emphasis Type="Italic"> Alw</Emphasis>26I,<Emphasis Type="Italic"> Eco</Emphasis>31I and<Emphasis Type="Italic"> Esp</Emphasis>3I,restriction endonucleases that recognise overlapping sequences

Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.     

Determination of methylation specificity of DsaV methyltransferase by a simple biochemical method.

We have developed a simple new method that can identify the base methylated by a sequence-specific DNA methyltransferase and have used it to identify the cytosine that is methylated by DsaV methyltransferase (M. DsaV) within its recognition sequence 5'-CCNGG. The method utilizes the fact that exonuclease III of E. coli does not degrade DNA ends with 3' overhangs and cannot hydrolyze a phosphorothioate linkage. DNA duplexes containing phosphorothioate linkages at specific positions were methylated with M. DsaV in the presence of [methyl-3H] S-adenosylmethionine and were subjected to exonuclease III digestion. The pattern of [methyl-3H] dCMP release from the duplexes was consistent with the methylation of the internal cytosine in CCNGG, but not of the outer cytosine. To establish the accuracy of this method, we confirmed the known specificity of EcoRII methyltransferase by the method. We also confirmed the specificity of M. DsaV using an established biochemical method that involves the use of a type IIS restriction enzyme. Methylation of CCWGG (W = A or T) sequences at the internal cytosines is native to E. coli and is not restricted by the modified cytosine restriction (Mcr) systems. Surprisingly, the gene for M. DsaV was significantly restricted by the McrBC system. We interpret this to mean that M. DsaV may occasionally methylate at sequences other than CCNGG or may occasionally methylate the outer cytosine in its recognition sequence.     

Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)

The properties and sources of all known class-I, class-II and class-III restriction endonucleases (ENases) and DNA modification methyltransferases (MTases) are listed and newly subclassified according to their sequence specificity. In addition, the enzymes are distinguished in a novel manner according to sequence specificity, cleavage position and methylation sensitivity. Furthermore, new nomenclature rules are proposed for unambiguously defined enzyme names. In the various Tables, the enzymes are cross-indexed alphabetically according to their names (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174, and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the ENases include relaxed specificities (integrated within Table II), the structure of the generated fragment ends (Table III), interconversion of restriction sites (Table IV) and the sensitivity to different kinds of DNA methylation (Table V). Table VI shows the influence of class-II MTases on the activity of class-II ENases with at least partially overlapping recognition sequences. Table VII lists all class-II restriction endonucleases and MTases which are commercially available. The information given in Table V focuses on the influence of methylation of the recognition sequences on the activity of ENases. This information might be useful for the design of cloning experiments especially in Escherichia coli containing M.EcodamI and M.EcodcmI [H16, M21, U3] or for studying the level and distribution of site-specific methylation in cellular DNA, e.g., 5'- (M)CpG-3' in mammals, 5'-(M)CpNpG-3' in plants or 5'-GpA(M)pTpC-3' in enterobacteria [B29, E4, M30, V4, V13, W24]. In Table IV a cross index for the interconversion of two- and four-nt 5'-protruding ends into new recognition sequences is complied. This was obtained by the fill-in reaction with the Klenow (large) fragment of the E. coli DNA polymerase I (PolIk), or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments [K56, P3].(ABSTRACT TRUNCATED AT 400 WORDS)     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared. A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases.     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.     

Cloning and analysis of the HaeIII and HaeII methyltransferase genes

The HaeIII methyltransferase (MTase) gene from Haemophilus aegyptius (recognition sequence: 5'-GGCC-3') was cloned into Escherichia coli in the plasmid vector pBR322. The gene was isolated on a single EcoRI fragment and on a single HindIII fragment. Clones carrying additional adjacent fragments were found to code also for the HaeII restriction endonuclease and HaeII modification MTase (recognition sequence: 5'-PuGCGCPy-3'). The sequence of the HaeIII modification gene was determined. The inferred amino acid sequence of the protein was found to share extensive similarity with other sequenced m5C-MTases. The central 'non-conserved' region of the M.HaeIII MTase, thought to form the nucleotide sequence-specificity domain, is almost identical to that of the M.BsuRI, M.BspRI and M.NgoPII MTases, which also recognize the sequence 5'-GGCC-3'.     

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

Two new restriction endonucleases from Proteus vulgaris.

Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.     

DNA site recognition and reduced specificity of the Eco RI endonuclease

It has been shown previously (Polisky, B., Green, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M., and Boyer, H. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3310-3314; Hsu, M., and Berg, P. (1978) Biochemistry 17, 131-138) that the cleavage sequence specificity of Eco RI endonuclease can be "relaxed" by various means. In this paper this phenomenon is explored in detail, in order to obtain further insight into the nature and selectivity of sequence recognition patterns between proteins and double-stranded nucleic acids. Using conditions of low ionic strength and alkaline pH, we have mapped the positions of potentially cleavable sites in the (completely sequenced) replicative form of the bacteriophage phi X174 genome, and have deduced their sequence. The time course of digestion of phi X174 DNA suggests that double-stranded sequences reading GGATTT, AAATTT, GAATTT, and GAATTA (only "top" strands, written 5' leads to 3', are shown) are cleaved readily under these conditions, while sequences reading CAATTN (N = A, T, G) resist attack. Cleavages at (at least) the more labile sites result in cohesive ends that are religatable. End group analysis of cleaved phi X174 DNA fragments indicates the presence of a 5'-terminal adenine residue on most of the fragments; some fragments may carry a 5'-terminal guanine residue, consistent with the cleavage site sequences suggested above. Addition of Mn2+ to cleavage reactions carried out at moderate salt concentrations and near-neutral pH induces the same pattern of cleavage seen at low ionic strength and alkaline pH. These results are combined with those from other studies, and are interpreted in terms of a model for the site-specific interaction of the Eco RI endonuclease with its substrate, considering both the effects of changes in DNA sequence and of environmental alterations. The resulting model is compared with data developed on similar grounds for Eco RI methylase (see Woodbury, C. P., Downey, R. L., and von Hippel, P. H. (1980) J. Biol. Chem. 255, 11526-11533), and attempts are made to define both common and differing molecular facets of the DNA recognition specificity of these companion (but genetically distinct) enzymes.     

Isolation and characterization of new restriction endonucleases from Vibrio parahaemolyticus: VpaK32I enzyme with the class-IIS heptanucleotide specificity, GCTCTTCN1/N4.

Six restriction endonucleases (ENases), classified into four different specificities, were found in a screen among 68 reference strains of Vibrio parahaemolyticus of human origin. Five of these ENases are isoschizomers of well-known ENases, while the remaining one, designated VpaK32I, is a novel and highly efficient class-IIS ENase with the hepatanucleotide recognition site, 5'-GCTCTTC(1/4)-3'.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.     

Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity

Restriction endonucleases have proven to be especially resistant to engineering altered substrate specificity, in part, due to the requirement of a cognate DNA methyltransferase for cellular DNA protection. The thermophilic restriction endonuclease BstYI recognizes and cleaves all hexanucleotide sequences described by 5'-R GATCY-3' (where R=A or G and Y=C or T). The recognition of a degenerate sequence is a relatively common feature of the more than 3000 characterized restriction endonucleases. However, very little is known concerning substrate recognition by such an enzyme. Our objective was to investigate the substrate specificity of BstYI by attempting to increase the specificity to recognition of only AGATCT. By a novel genetic selection/screening process, two BstYI variants were isolated with a preference for AGATCT cleavage. A fundamental element of the selection process is modification of the Escherichia coli host genomic DNA by the BglII N4-cytosine methyltransferase to protect AGATCT sites. The amino acid substitutions resulting in a partial change of specificity were identified and combined into one superior variant designated NN1. BstYI variant NN1 displays a 12-fold preference for cleavage of AGATCT over AGATCC or GGATCT. Moreover, cleavage of the GGATCC sequence is no longer detected. This study provides further evidence that laboratory evolution strategies offer a powerful alternative to structure-guided protein design.     

Alw26I, Eco31I and Esp3I--type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA.

The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.     

Expanding the recognition of the minor groove of DNA by incorporation of beta-alanine in hairpin polyamides

In order to expand the recognition code by hairpin polyamides to include DNA sequences of the type 5'-CWWC-3' two polyamides, PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) and PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) were synthesized which have in common an Py/Im pair in the terminal position for targeting C x G but differ with respect to internal placement of a beta-alanine residue. The equilibrium association constants (Ka) were determined at four DNA sites which differ at a single common position, 5'-TNTACA-3' (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that the eight-ring hairpin PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) binds the four binding sites with similar affinities, Ka = 1.3-1.9 x 10(10) M(-1) indicating that there is no preference for the position N. In contrast, a redesigned polyamide PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) that places an internal flexible aliphatic beta-alanine to the 5'-side of a key imidazole group bound the match site 5'-TCTACA-3' with high affinity and good sequence discrimination (Ka(match) = 4.9 x 10(10) M(-1) and the single base pair mismatch sites with 5- to 25-fold lower affinity). These results expand the repertoire of sequences targetable by hairpins and emphasize the importance of beta-alanine as a key element for minor groove recognition.     

The synthesis and properties of oligodeoxyribonucleotides containing N6-methoxyadenine.

Oligodeoxyribonucleotides containing N6-methoxyadenine (M) have been synthesized. The order of stability of duplexes consisting of synthesized oligodeoxyribonucleotides, 5'd(CCTGGTAXCAGGTCC)3'-5'd(GGACCTGNTACCAGG)3' (X = M, A, G. N = A, G, T, C), was M: A (Tm = 52 degrees C) greater than M: T (50 degrees C) greater than M: G (48 degrees C) greater than M: C (46 degrees C) observed by thermal denaturation in a buffer of 0.01 M Na cacodylate, and 0.1 M NaCl at pH 7.0. The Tms are within a range of 6 degrees of difference, which is smaller than those of Tms of the duplexes containing A:N pairs (11 degrees) and G:N pairs (11 degrees). DNA replication study on a template-primer system, 5'd(32p-CAGCTTTCGC)3' 3'd(GTCGAAAGCGMAGTCG)5', showed that TTP and dCTP were incorporated into DNA strands at a site opposite to M by Klenow DNA polymerase, but dATP and dGTP were not.     

Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy

The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.