Studies on sequence recognition by type II restriction and modification enzymes

Type II DNA restriction and modification systems are ideally suited for analysis of mechanisms by which proteins specifically recognize unique DNA sequences. Each system is comprised of a unique DNA recognition site and two enzymes, which in those cases examined in detail, are comprised of distinct polypeptide chains. Thus, not only are the DNA substrates extremely well defined, but each system affords the opportunity to compare distinct proteins which interact with a common DNA sequence. This review will focus only on those Type II systems which have been examined in sufficient molecular detail to permit some insight into modes of specific DNA-protein interaction.     

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.     

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes.

The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.     

A computer algorithm to determine the recognition site of restriction enzymes

A new algorithm is proposed to determine the type-II restrictionendonucleases' recognition site knowing the digested DNA sequenceand fragment lengths in an actual case. The algorithm is implementedfor the Commodore 64 microcomputer. Received on January 6, 1987; accepted on June 19, 1987     

Conservation of complex DNA recognition domains between families of restriction enzymes

One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes. Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences. The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence. We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence. We have determined the sequence recognized by EcoE, a member of the A family. This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family. We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family. There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE. A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence.     

KpnI restriction endonuclease and methyltransferase exhibit contrasting mode of sequence recognition

The molecular basis of the interaction of KpnI restriction endonuclease (REase) and the corresponding methyltransferase (MTase) at their cognate recognition sequence is investigated using a range of footprinting techniques. DNase I protection analysis with the REase reveals the protection of a 14–18 bp region encompassing the hexanucleotide recognition sequence. The MTase, in contrast, protects a larger region. KpnI REase contacts two adjacent guanine residues and the single adenine residue in both the strands within the recognition sequence 5′-GGTACC-3′, inferred by dimethylsulfate (DMS) protection, interference and missing nucleotide interference analysis. In contrast, KpnI MTase does not show elaborate base-specific contacts. Ethylation interference analysis also showed the differential interaction of REase and MTase with phosphate groups of three adjacent bases on both strands within the recognition sequence. The single thymine residue within the sequence is hyper- reactive to the permanganate oxidation, consistent with MTase-induced base flipping. The REase on the other hand does not show any major DNA distortion. The results demonstrate that the differences in the molecular interaction pattern of the two proteins at the same recognition sequence reflect the contrasting chemistry of DNA cleavage and methylation catalyzed by these two dissimilar enzymes, working in combination as constituents of a cellular defense strategy.     

Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme

We have studied the importance of charge and hydrogen-bonding potential of the phosphodiester backbone for binding and cleavage by EcoRI restriction endonuclease. We used 12-mer oligodeoxynucleotide substrates with single substitutions of phosphates by chiral methylphosphonates at each position of the recognition sequence -pGpApApTpTpCp-. Binding was moderately reduced between 4- and 400-fold more or less equally for the R(P) and S(P)-analogues mainly caused by missing charge interaction. The range of cleavage effects was much wider. Four substrates were not cleaved at all. At both flanking positions and in the purine half of the sequence up to the central position, cleavage was more impaired than binding and differences between R(P) and S(P) diastereomeres were more pronounced. These effects are easily interpreted by direct phosphate contacts seen in the crystal structure. For the effects of substitutions in the pyrimidine half of the recognition sequence, more indirect effects have to be discussed.     

Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes

Background

Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances.

Scope of review

This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family.

Major conclusions

It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid–base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions.

General significance

Glutathione-dependent enzymes are excellent study objects for structure–function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

Asymmetric photocross-linking pattern of restriction endonuclease EcoRII to the DNA recognition sequence

The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.     

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

The interaction of MvaI restriction endonuclease with 14-membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m5dC for the internal dC residue, as well as substitution of fl5dU or rU for dT did not influence the initial rate of hydrolysis (v0) of modified strands, whereas the hydrolysis of unmodified strands was inhibited in some cases. Furthermore, the substitution of a pyrophosphate bond for a scissile phosphodiester bond in one strand completely inhibited digestion in this strand without any decrease of the rate of hydrolysis of the unmodified strand. In contrast to EcoRII endonuclease, which recognizes the same DNA sequence, in the case of MvaI endonuclease substrate recognition is possible in a wide range of conformational, electronic and hydrophobic alterations within the recognition site.     

Codification and evolution of experimentally observed specific recognition sites for restriction enzymes on DNA

The list of published restriction endonucleases along with their substrates provides an excellent data base for the evaluation of the evolution and codification of the key elements for specific recognition sites on the DNA. In this paper the considerations will be limited to palindromic tetramer-, pentamer-, and hexamer-sequences. It is basically assumed that each base pair within these sequences has to be recognized by directionally unique bidentate hydrogen bonds either within the plane of the base pair or by bridging the appropriate H-bond donor/acceptor groups of the neighbouring bases of the same strand. Thus sequence specificity is mediated by twelve (eight) H-bonds, originating from the protein recognition modules. Besides a pronounced preference for GC base pairs expressed by their high frequency in the most abundant sequences, serving the need of maximal thermodynamic stability of the double helical substrates, it can also be shown that the stacking of consecutive bases within the recognition site sequences plays a major role in shaping the particular DNA/protein interface. Finally it will be demonstrated that the full set of sequences discussed in this paper can readily be derived by stepwise expanding the vocabulary of three simple tetrameric sequences by inserting single base pairs into the centre of a minimal sequence, thus creating all the published pentameric restriction sites, or by inserting/adding two GC base pairs in a palindromic way, thus creating the known multiplicity of hexameric sites.     

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.     

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.     

Identification of seven species of hymenopteran parasitoids of Spodoptera frugiperda, using polymerase chain reaction amplification and restriction enzyme digestion

1 The fall armyworm Spodoptera frugiperda is a voracious pest of numerous crops of economic importance throughout the New World. In its native Mexico, larvae can be attacked by several species of parasitic wasps, which are candidate biological control agents against this and other lepidopteran pests. 2 We attempted to survey the parasitoid fauna on S. frugiperda in maize and sorghum fields throughout Mexico. However, our efforts have been hampered by the incomplete development of parasitoid larvae emerging from collected Spodoptera caterpillars. 3 This problem was solved by developing a method to identify seven species of parasitic wasps using polymerase chain reaction amplification and restriction enzyme digestion. This enables the precise determination of the species of those parasitoid larvae that are usually not morphologically identifiable.     

One oxidant,many pathways: a theoretical perspective of monooxygenation mechanisms by cytochrome P450 enzymes

Density functional theoretical studies of monooxygenation reactivity of the high-valent oxoiron(IV) porphyrin cation-radical compound of cytochrome P450, the so-called Compound I, and of its precursor, the ferric(III)-hydroperoxide species, are described. The degeneracy of the spin states of Compound I, its electron deficiency, and dense orbital manifold lead to two-state and multi-state reactivity scenarios and may thereby create reactivity patterns as though belonging to two or more different oxidants. Most of the controversies in the experimental data are reconciled using Compound I as the sole competent oxidant. Theory finds ferric(III)-hydroperoxide to be a very sluggish oxidant, noncompetitive with Compound I. If and when Compound I is absent, P450 oxidation will logically proceed by another form, but this has to be more reactive than ferric(III)-hydroperoxide. Theoretical studies are conducted to pinpoint such an oxidant for P450.

Evolution of sequence recognition by restriction-modification enzymes: selective pressure for specificity decrease

Several type II restriction-modification (RM) gene complexes kill host bacterial cells that have lost them, through attack on the chromosomal recognition sites of these cells. Two RM gene complexes recognizing the same sequence cannot simultaneously enjoy such stabilization through postsegregational host killing, because one will defend chromosomal sites from attack by the other. In the present work, we analyzed intrahost competition between two RM gene complexes when the recognition sequence of one was included in that of the other. When the EcoRII gene complex, recognizing 5'-CCWGG (W = A, T), is lost from the host, the SsoII gene complex, which recognizes 5'-CCNGG (N = A, T, G, C), will prevent host death by protecting CCWGG sites on the chromosome. However, when the SsoII (CCNGG) gene complex is lost, the EcoRII (CCWGG) gene complex will be unable to prevent host death through attack by SsoII on 5'-CCSGG (S = C, G) sites. These predictions were verified in our experiments, in which we analyzed plasmid maintenance, cell growth, cell shape, and chromosomal DNA. Our results demonstrate the presence of selective pressure for decrease in the specificity of recognition sequence of RM systems in the absence of invading DNA.