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.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

Analysis of a drosophila tRNA gene cluster: two tRNALeu genes contain intervening sequences

A recombinant DNA phage containing a cluster of Drosophila melanogaster tRNA genes has been isolated and analyzed. The insert of this phage has been mapped by in situ hybridization to chromosomal region 50AB, a known tRNA site. Nucleotide sequencing of the entire Drosophila tRNA coding region reveals seven tRNA genes spanning 2.5 kb of chromosomal DNA. This cluster is separated from other tRNA regions on the chromosome by at least 2.7 kb on one side, and 9.6 kb on the other. Two tRNA genes are nearly identical and contain intervening sequences of length 38 and 45 bases, respectively, in the anticodon loop. These two genes are assigned to be tRNALeu genes because of significant sequence homology with yeast tRNA3Leu, and secondary structure homology with yeast tRNA3Leu intervening sequence. In addition, an 8 base sequence (AAAAUCUU) is conserved in the same location in the intervening sequences of Drosophila tRNALeu genes and a yeast tRNA3Leu gene. Similar sequenes occur in all other tRNAs containing intervening sequences. The remaining five genes are identical tRNAIle genes, which are also identical to a tRNAIle gene from chromosomal region 42A. The 5' flanking regions are only weakly homologous, but each set of isoacceptors contains short regions of strong homology approximately 20 nucleotides preceding the tRNA coding sequences: GCNTTTTG preceding tRNAIle genes; and GANTTTGG preceding tRNALeu genes. The genes are irregularly distributed on both DNA strands; spacing regions are divergent in sequence and length.     

Creating seamless junctions independent of restriction sites in PCR cloning

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5′-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine (^m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5′ overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.     

Fast analysis of DNA and protein sequence on Apple IIe: restriction sites search, alignment of short sequence and dot matrix analysis.

A fast restriction sites search algorithm using a quadruplet look-ahead feature has been written in 6502 assembly language code. The search time, tested on the sequence of pBR322, is 4.1 s/kilobase using a restriction site library including 112 specificities corresponding to a total site length of over 700 bases. The search for a short sequence (less than 36 bases) within a longer one (up to 9999 bases) with a given number of mismatches or gaps allowed has also been written in assembly language. Typical run time for the search of a 12 base sequence with 1, 2 or 3 gaps allowed are 6.2, 9.4 or 13.6 s/kilobase, respectively. The dot matrix analysis needs 7.5 minutes per square kilobase when using a stringency of 15 matched bases out of 25. A 7/21 matrix of two 500 amino acid proteins is obtained in 3 minutes. These three routines are included in DPSA, a general package of programs allowing manipulation and analysis of DNA and protein sequences.     

An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other Type IIP enzyme reported to date, an MspI monomer and not a dimer binds to a palindromic DNA sequence. The enzyme makes specific contacts with all 4 base pairs in the recognition sequence, by six direct and five water-mediated hydrogen bonds and numerous van der Waal contacts. This MspI-DNA structure represents the first example of asymmetric recognition of a palindromic DNA sequence by two different structural motifs in one polypeptide. A few possible pathways are discussed for MspI to cut both strands of DNA, either as a monomer or dimer.     

Specific minor groove solvation is a crucial determinant of DNA binding site recognition

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein''s ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein.     

Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA.

Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of trans- acting factors. One such HeLa cell factor, EBP1, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human beta-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBP1 binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding.     

Recognition sequence for the restriction endonuclease BglI from Bacillus globigii

Restriction endonuclease BglI recognizes the DNA sequence (Formula: see text) and cleaves each strand at the site indicated, thus generating 3' protruding ends. The recognition sequence was deduced by correlating mapping data with nucleotide sequence information and the position of cleavage was unambiguously determined by 32P labeling of 5' termini produced by BglI digestion.     

Additional Restriction Endonuclease Cleavage Sites on the Bacteriophage P22 Genome

We present complete restriction endonuclease cleavage site maps of the bacteriophage P22 chromosome for 16 enzymes with six base recognition sequences, thereby positioning 116 new sites on the chromosome. Twenty-four such restriction maps for P22 DNA, containing 162 sites, have now been completed, and three enzymes were found that did not cut P22 DNA. Our results are consistent with the ideas that ClaI does not cleave the methylated recognition sequence ATCGA(me)T or A(me)TCGAT and StuI does not cleave the methylated recognition sequence AGGCC(me)T.     

The sequence of the chromosomal mouse β-globin major gene: Homologies in capping,splicing and poly(A) sites

We have determined the entire nucleotide sequence of a cloned β-globin^maj gene derived from the BALB/c mouse. This sequence is 1567 bases long and includes the 5′ cap region as well as the presumptive poly(A) addition site of β-globin mRNA. The sequence establishes the fact that the gene is encoded in three discontinuous segments of DNA interrupted by two intervening sequences and precisely locates each. The smaller intervening sequence, 116 bases long, occurs between Arg and Leu codons at codon positions 30 and 31. The larger intervening sequence of 646 bases also occurs between Arg and Leu codons, but at codon positions 104 and 105. There is striking homology between the borders of the two intervening sequences, but no extensive dyad symmetry. Furthermore, the DNA region that just precedes and overlaps the 5′ cap structure of the mRNA shows homology to corresponding regions in other eucaryotic genes including the late adenovirus promoter. The 3′ untranslated sequence is closely homologous to that of the rabbit β-globin mRNA. The sequence thus allows us to identify several noncoding regions of potential importance for the expression and processing of genetic information. It also provides a basis for future comparison with other sequenced genes and a defined substrate for the development of direct tests of gene function.     

Rational engineering of sequence specificity in R.MwoI restriction endonuclease

R.MwoI is a Type II restriction endonucleases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 5'-GCNNNNNNNGC-3' (where N indicates any nucleotide), and hydrolyzes the phosphodiester bond in the DNA between the 7th and 8th base in both strands. R.MwoI exhibits remote sequence similarity to R.BglI, a REase with known structure, which recognizes an interrupted palindromic target 5'-GCCNNNNNGGC-3'. A homology model of R.MwoI in complex with DNA was constructed and used to predict functionally important amino acid residues that were subsequently targeted by mutagenesis. The model, together with the supporting experimental data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinformatics analysis, we designed substitutions of the S310 residue in R.MwoI to arginine or glutamic acid, which led to enzyme variants with altered sequence selectivity compared with the wild-type enzyme. The S310R variant of R.MwoI preferred the 5'-GCCNNNNNGGC-3' sequence as a target, similarly to R.BglI, whereas the S310E variant preferentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleotide residues. Our results represent a case study of a REase sequence specificity alteration by a single amino acid substitution, based on a theoretical model in the absence of a crystal structure.     

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

The ovalbumin gene: alleles created by mutations in the intervening sequences of the natural gene.

Two allelic forms of the natural chicken ovalbumin gene have been independently cloned. These alleles differ from each other by an Eco RI restriction cleavage site in one of the seven intervening sequences within the natural ovalbumin gene. Restriction endonuclease mapping and sequence analyses of these cloned genotypic alleles have shown identical sequence organization and molecular structures of the interspersed structural and intervening sequences except for the particular Eco RI cleavage site. Sequencing data of the cloned DNA suggest that this Eco RI site may be created or eliminated by a single base mutation in the intervening sequence of the ovalbumin gene. The occurrence of apparent homozygous and heterozygous allelic forms of the ovalbumin gene in individual hens and roosters within the same breed has been observed. 10 and 40% of the chickens examined are homozygous for the ovalbumin gene with and without the extra Eco RI site, respectively, while 50% of them are heterozygous. Further analysis of individual chicken DNA cleaved by restriction endonuclease Hae III has revealed that there may be a series of such mutational variations within the ovalbumin gene. We have identified two Hae III cleavage sites that do not occur in all of the chickens, thus giving rise to several additional allelic variations of the ovalbumin gene. At least one of these Hae III sites is situated in the intervening sequence of the ovalbumin gene, and its lcoation has been mapped. Such allelic variations must be taken into consideration when determining eucaryotic gene structure by restriction mapping of the genomic DNA. Furthermore, this type of mutation within the intervening sequences of an eucaryotic gene has no known phenotypic manifestation. It represents an extrastructural silent mutation that must be taken account of in studies to estimate the rates of eucaryotic gene sequence divergence during evolution.     

New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila.

Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation. In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila. Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites. They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs. This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs. In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G. When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs. This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion. These results suggest a strong mechanistic link between chromosome breakage and telomere formation.     

Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease.     

Patterns of lambda Int recognition in the regions of strand exchange

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for lambda contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.     

Predictive motifs derived from cytosine methyltransferases.

Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.