what does an enzyme do to a dna molecule

• Periodical List
• EMBO Rep
• 5.2(4); 2001 Apr 15
• PMC1083870

EMBO Rep. 2001 Apr 15; 2(iv): 271–276.

Enzymes that go on Dna under control

Meeting: DNA enzymes: structures and mechanisms

Alfred Pingoud

^aneInstitut für Biochemie, Justus-Liebig-Universität, 35392 Giessen, Federal republic of germany,

Albert Jeltsch

^oneInstitut für Biochemie, Justus-Liebig-Universität, 35392 Giessen, Germany,

Anthony Maxwell

²Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH,

David Sherratt

^threeSection of Biochemistry, University of Oxford, Oxford OX1 3QU, UK

Received 2001 Jan 22; Revised 2001 Feb 21; Accepted 2001 Feb 27.

Introduction

Life demands not only the true-blue and controlled replication of DNA but, in addition, many other enzymatic processes involving Dna, including topoisomerase activeness, recombination, repair, restriction and modification. These topics and their interrelationships were discussed at the IUBMB symposium in Bangalore (India) on DNA enzymes: structures and mechanisms (December one–iii, 2000), which was organized by 5. Nagaraja and D.Due north. Rao (Bangalore, India) and brought together about 200 scientists from all over the globe. Whereas most presentations focused on detailed biochemical and genetic mechanisms, several reminded united states that enzymes and Deoxyribonucleic acid are components of complex living organisms that live, dice and evolve.

Topoisomerases and DNA replication

During replication, rapid and accurate unlinking of duplex DNA is required, which is carried out by 3 classes of motor proteins: helicases, topoisomerases and condensins (reviewed in Wang, 1996, 1998; Holmes and Cozzarelli, 2000). The roles of topoisomerases in replication fork progression in Escherichia coli were illustrated by Northward. Cozzarelli (Berkeley, CA). He described microarray experiments showing that inhibition of DNA gyrase leads to a slow abort of replication, whereas inhibition of both gyrase and DNA topoisomerase (topo) Four results in rapid abort, supporting a function for topo Iv in the removal of positive supercoils that accrue in front of the replication fork. In vitro experiments, in particular studies using single molecule enzymology, show that topo IV preferentially relaxes positively supercoiled Dna.

The enzymology of Dna gyrase was discussed past A. Maxwell (Norwich, UK) and A. Bates (Liverpool, UK). Gyrase is the only topoisomerase that tin introduce negative supercoils into DNA. Its mechanism involves the ATP-driven capture of one segment of DNA, the T-segment, which is passed through another segment, the G-segment, bound to the enzyme (Figure ​ 1). Proof that the T-segment passes through the enzyme was provided by protein cross-linking across the subunit interfaces of the enzyme. Recent data back up a model in which the T-segment is captured irrespective of the topological state of the Dna; this sets up an 'on-enzyme equilibrium' with the T-segment bound in the interior of the enzyme. The superhelical density of the DNA and the gratuitous energy of ATP hydrolysis decide the subsequent passage of the T-segment through the Thousand-segment, a hypothesis supported by theoretical calculations.

Fig. 1. Model for strand passage past DNA gyrase. The 43- and 47-kDa domains of GyrB are represented in xanthous and as green circles, respectively. GyrA is represented in ruby; the 33-kDa domain is omitted for clarity. In this model, gyrase binds to Deoxyribonucleic acid (blue; ane) and wraps ∼130 bp around the poly peptide (but one arm of the wrap is shown for clarity). The spring DNA (the 1000 segment) is cleaved (2) and another office of the wrap (the T segment) is captured by the 43-kDa domains of GyrB, post-obit ATP (filled triangle) bounden (3). This sets upwards the on-enzyme equilibrium of the T segment (dashed arrow) that tin lead to strand passage and DNA supercoiling.

Whereas gyrase and topo IV are type Two topoisomerases that transiently innovate double-strand breaks into Dna, type I enzymes catalyze transient single-strand breaks. Bacterial type I enzymes (type IA) make covalent bonds to the five′-phosphate of DNA during their reactions, whereas eukaryotic enzymes (blazon IB) form covalent linkages to the 3′-phosphate. V. Nagaraja (Bangalore, India) discussed aspects of topo I from Mycobacterium smegmatis. This enzyme is unusual in that, in contrast to almost other topoisomerases, it binds Deoxyribonucleic acid site-specifically. In addition, information technology is stimulated by single-stranded binding (SSB) protein. M.-A. Bjornsti (Memphis, TN) discussed topo I from yeast. Eukaryotic type I enzymes are targets for the anti-tumour drug camptothecin, which stabilizes the covalent enzyme-DNA adduct. Its cytotoxicity is thought to be a issue of the collision of the Dna replication fork with the topoisomerase-drug complex on DNA. Bjornsti'due south lab has developed a yeast screen to define cellular components involved in processing the stalled complexes. 2 proteins, Doa4p (a ubiquitin hydrolase) and Sla1p (a component of the cortical actin cytoskeleton) have been implicated in the response to topoisomerase-induced DNA damage. How these proteins are involved in this response is unclear at nowadays.

Other enzymes involved in DNA replication include the Deoxyribonucleic acid helicases. J. Gowrishankar (Hyderabad, India) presented work on Escherichia coli UvrD, a 3′–5′ helicase. He showed that uvrD zero mutants are incompatible with lon mutations, equally a consequence of chronic low-level induction of the SOS response. Based on this and other work, it was proposed that the UvrD helicase participates in lagging strand replication, specifically in removing secondary construction.

An important trouble in Deoxyribonucleic acid metabolism is that cytosines are easily converted to uracils by deamination. In fact, this is the most common promutagenic lesion. B. Connolly (Newcastle, United kingdom of great britain and northern ireland) explained that uracil-DNA glycosylases, which remove the uracil from damaged DNA, appear to be absent in archaea. Even so, DNA polymerases from some archaea (like the Vent and Pfu polymerases) can specifically recognize dU in the template strand approximately four bases from the primer-template junction. This results in stalling of the polymerase and may exist the first pace in the repair pathway.

S. Hasnain (Hyderabad, India) discussed replication origins in baculovirus. These viruses have multiple origins and 1 of these, hr1, acts not merely as an ori merely also equally a transcriptional enhancer, suggesting cross-talk between the processes of replication and transcription. Due south. Bhattacharya (New Dehli, India) showed that multiple replication origins are also a feature of the ribosomal RNA genes of the parasite Entamoeba histolytica, which are located on high copy number extrachromosomal circular Deoxyribonucleic acid molecules in this organism. D. Bastia (Durham, MA) discussed the mechanism of replication termination in bacteria and yeast. In many systems, this process occurs following the arrest of replication forks at sequence-specific termini and involves a termination poly peptide (Tus in E. coli, RTP in Bacillus subtilis). Study of the termination of replication of rDNA of Saccharomyces cerevisiae shows that point mutations in the fob (fork blocking less) factor abolish fork abort and extend the life-span of the cell. The Play a trick on protein is thought to interact with a Dna-bounden protein that binds at the replication terminus.

Restriction-modification

Brake-modification (RM) systems are found ubiquitously in the prokaryotic kingdom where they serve every bit defense systems against strange Dna. Currently, 47 type I, 3320 type Ii and eight blazon Three systems are known. R. Roberts (Beverly, MA) pointed out that, until recently, brake enzymes had been identified by obtaining bacteria from culture collections and ecology samples and analyzing them for brake activeness. Now information technology is possible to screen new DNA sequences for DNA methyltransferase (MTase) genes, which tin can be identified by their conserved motifs, and and then looking for associated genes. These are proficient candidates for restriction endonuclease genes, because the genes of Deoxyribonucleic acid methyltransferases and restriction endonucleases are often linked. Recent genome projects take identified an unexpectedly large number of putative RM systems in this manner. For case, 25 different MTase genes were identified in the Helicobacter pylori genome, and some of these were associated with brake enzyme genes. Screening databases may, therefore, become a very productive method of finding restriction enzymes with new specificities. A. Piekarowicz (Warsaw, Poland) reported on ane instance of such an approach that led to the identification of a new type IC brake enzyme, NgoAXVI.

Blazon I and type III restriction enzymes, equally well as the methyl-dependent McrBC enzyme, require two recognition sites and depend on ATP (McrBC: GTP) hydrolysis for DNA cleavage (reviewed in Rao et al., 2000). Cleavage occurs when two such enzymes collide while translocating DNA, which explains the requirement for ii sites (Effigy ​ 2). T. Bickle (Basel, Switzerland) demonstrated that blazon I enzymes and McrBC can as well be stimulated to cleave Deoxyribonucleic acid when they run across a non-specific cake. Type III enzymes, in contrast, are inhibited past such blocks, because each translocating enzyme cuts but one strand and requires the cooperation of another enzyme for cleavage of both DNA strands.

Fig. 2. Model for the activation of brake enzymes that crave ATP (or GTP) for DNA cleavage. After binding of two enzyme molecules (or complexes) to two recognition sites on a linear DNA molecule, the DNA is translocated in an active, energy dependent process which requires ATP or GTP, depending on the organisation. Thereby, the DNA is looped out. Cleavage occurs after collision of two translocating complexes, at random sites in the vicinity of the recognition sites (type III enzymes and McrBC) or further away from them (blazon I enzymes).

Although most type 2 brake enzymes are homodimers that interact with ane copy of their palindromic recognition site, subtypes be that crave cooperation of ii sites (reviewed in Pingoud and Jeltsch, 1997). As pointed out by South. Halford (Bristol, UK), this is the instance not only for blazon IIe enzymes, like NaeI, but too for type IIf enzymes, like SfiI, and type IIS enzymes, like FokI. Type IIf enzymes are homotetramers with two separate Dna binding sites, each formed by two subunits. SfiI is only active when both Deoxyribonucleic acid-bounden sites are occupied, leading to a simultaneous cleavage of both sites. For FokI, which consists of a cleavage and a recognition domain, information technology was shown previously that dimerization of the enzyme on the Deoxyribonucleic acid is required for cleavage to occur. Halford has now shown that two recognition sites are required for efficient cleavage, because each of the recognition domains must interact with a recognition sequence. All iii types of enzymes act optimally with two sites on the same Dna, where they trap the Dna between the sites in a loop.

Singular type II brake enzymes continue to be discovered. BbvCI is a heterodimeric restriction enzyme that recognizes an disproportionate sequence. The subunits are inactive individually, merely active together. This makes it possible to create specific nicking enzymes past inactivating one subunit using site-directed mutagenesis, every bit was reported by G. Wilson (Beverly, MA). A. Janulaitis (Vilnius, Lithuania) obtained a like effect for the heterodimeric Bpu10I restriction enzyme. He also succeeded in relaxing the substrate specificity of Eco57I, a monomeric restriction and modification enzyme, which has a single target recognition domain. In this study, random mutagenesis was used to generate a variant that interacts not but with the approved CTGAAG site, only also with CTGGAG sites. The molecular basis of this relaxed specificity has yet to be determined. 5. Siksnys (Vilnius, Lithuania) reported the discovery of a new type IIs restriction enzyme, BfiI, which is not dependent on divalent metal ions for cleavage. This enzyme shows sequence similarity to a not-specific nuclease from Salmonella typhimurium and presumably uses a like catalytic mechanism.

A. Pingoud (Giessen, Germany) discussed the mechanism of Deoxyribonucleic acid cleavage by type II restriction enzymes and homing endonucleases, which share a mutual role, simply in full general have different structures and presumably follow different mechanisms of cleavage. In spite of the fact that detailed construction information is available for many restriction enzymes that share a common catalytic motif, there is no consensus regarding the catalytic mechanism or the number of Mgⁱⁱ⁺ ions that are involved in catalysis. In principle, the same is true for other phosphoryl transferases with a brake enzyme-like catalytic centre. Examples of such proteins are the Vsr repair enzyme (E. coli) and the Hjc resolvase (Pyrococcus furiosus). The crystal structure of the latter was presented by K. Morikawa (Osaka, Japan) (Effigy ​ three). In dissimilarity, the mechanism of DNA cleavage by the homing endonucleases of the HNH family, due east.chiliad. I-PpoI seems to be meliorate established, because structural information is available for the free enzyme every bit well every bit enzyme-substrate and enzyme-product complexes, in add-on to detailed biochemical information for this and related enzymes of the 'ββα-Me finger' superfamily. These enzymes require a Mg²⁺ ion as cofactor which is bound to a conserved Asn. The attacking hydroxyl ion is generated by a conserved His, transition state stabilization involves a conserved Arg and leaving group protonation is afforded by a water molecule from the hydration sphere of the Mg²⁺ ion.

Fig. three. Comparing of the brake endonuclease folds of Hjc and Vsr as an example of conservation of structures for enzymes with related functions. The ribbon diagrams of Hjc and Vsr are shown in the same orientation after superimposition of the two structures. The side chains of the agile site residues are highlighted, and the two conserved Asp residues are labeled. This figure was kindly provided by T. Nishino and G. Morikawa.

Leaner have adult RM systems to fight bacteriophages. These in turn have developed diverse means of escaping restriction past bacterial RM systems. D. Dryden (Edinburgh, UK) reported the results of a biochemical analysis of the gene 0.3 protein from bacteriophage T7, which is an inhibitor of type I restriction-modification enzymes. This poly peptide binds stoichiometrically to the restriction enzyme and, because of its elongated form and negative surface charge, presumably completely fills the DNA binding site of the enzyme and thereby prevents DNA bounden.

RM systems consist of restriction endonucleases and DNA methyltransferases (MTases) (reviewed in Cheng, 1995; Robertson and Wolffe, 2000). Even so, since the blueprint of Deoxyribonucleic acid methylation adds information to the Dna and thereby extends its coding capacity, MTases are not only the companions of restriction enzymes in RM systems, but have many other vital functions. In prokaryotes, they play roles in DNA repair and the regulation of gene expression and DNA replication. In eukaryotes, Deoxyribonucleic acid methylation more often than not leads to transcriptional silencing of genes. It contributes to epigenetic processes such as X-chromosome inactivation, imprinting and cistron regulation. With the discovery that several DNA MTases are essential for development in mice, the importance of Dna methylation has become widely accustomed.

X. Cheng (Atlanta, GA) presented the structure of the Dnmt2 protein, which could be the kickoff structure of a eukaryotic Deoxyribonucleic acid MTase. The protein has an MTase fold and possesses all of the characteristic catalytic motifs, but seems to be devoid of any catalytic activity. This raises questions such as whether it is indeed an enzyme and what its substrate actually is (DNA, RNA or something else). Further discussions on eukaryotic enzymes past Cheng and A. Jeltsch (Giessen, Germany) dealt with the Dnmt1, Dnmt3a and Dnmt3b enzymes. Both speakers reported results obtained with truncated proteins and isolated domains of these huge enzymes (comprising up to 1700 amino acid residues), as well as presenting results apropos the enzymatic assay of purified enzymes. Since the catalytic domain of Dnmt1 (∼500 amino acrid residues) is not active in isolated form, it must be nether tight control of other parts of the enzyme. This interaction might be required to ensure a high specificity for hemimethylated Dna. In spite of the progress in the field, the process of Dna methylation in eukaryotes, in particular the mechanisms that create the blueprint of DNA methylation, is still poorly understood.

Somewhat more is known about Deoxyribonucleic acid methylation in prokaryotes. R. Gumport (Urbana, IL) presented the structure of the prokaryotic Thou.RsrI MTase, confirming conjecture that the catalytic domains of all MTases share a common architecture. S. Klimasauskas (Vilnius, Lithuania), D.N. Rao (Bangalore, India), Gumport and Jeltsch discussed the molecular enzymology of four prokaryotic MTases (M.HhaI, M.EcoP15, M.RsrI and Thousand.EcoRV). The catalytic bicycle of these enzymes involves DNA and cofactor binding, target site location and recognition, and conformational changes of the circuitous including base flipping and methyl grouping transfer (Effigy ​ iv). Differences in detail became apparent; due east.k. the club of substrate and cofactor binding and the rate-limiting step differs in various MTases. two-aminopurine proved to exist a good tool for analyzing the kinetics of the conformational changes that the Deoxyribonucleic acid undergoes in complex with MTases, including base flipping.

Fig. 4. Mechanism of target site location by brake endonucleases and DNA methyltransferases. Beginning Dna is leap non-specifically, then the target site is located by facilitated diffusion (sliding and hopping) on the DNA. Contacts to the target sites induce conformational changes of the enzyme and the DNA which in turn trigger catalysis. This mechanism is common amid most enzymes that specifically collaborate with Dna.

Deoxyribonucleic acid recombination and repair

The last session of the meeting was devoted to recombination and repair, processes essential for generating genetic diversity and maintaining the DNA integrity (reviewed in Kuzminov, 1999). D. Sherratt (Oxford, Great britain) described the inter-relationships between DNA replication and homologous recombination, site-specific recombination, chromosome segregation and jail cell sectionalization in Eastward. coli. When DNA replication forks encounter DNA damage or stalled transcription complexes, they frequently stall or may even break. Productive replication forks tin reform by the procedure of homologous recombination. In bacteria with circular chromosomes this can pb to the formation of chromosomal dimers which cannot exist segregated to daughter cells at prison cell division. 2 processes act to minimize the trouble generated by crossover events. Showtime, homologous recombination is biased so that non-crossover events predominate over crossovers. This bias requires the action of the Ruv proteins. Secondly, whatever dimers formed are converted to monomers by the XerCD site-specific recombination system. The XerCD recombinases participate in a highly coordinated nucleoprotein molecular motorcar that completes a recombination reaction just when ∼thirty bp recombination sites, termed dif, are present in chromosome dimers at the time of cell division. The reaction is controlled temporally and spatially by the localization of the recombination machine to the partitioning septum as information technology forms during cell division; just correctly positioned dif sites within dimers can access this part of the recombination machine in the septum.

XerCD belongs to the tyrosine recombinase family unit of site-specific recombinases, which are structurally and mechanistically related to the type IB topoisomerases of eukaryotes. Farther mechanistic insight into the reaction mechanism of these enzymes was provided past new results on the Flp recombinase from Southward. cerevisiae (M. Jayaram, Austin, TX). Iv molecules of Flp mediate a site-specific recombination reaction between 2 ∼30 bp frt sites. Once two frt sites have been synapsed by Flp–Flp interaction, the recombination reaction occurs by two pairs of strand exchanges that are separated in time and space. A Holliday junction (HJ)-containing molecule is a reaction intermediate. Topological analysis demonstrated that the recombination sites align in an antiparallel sense with respect to each other and that strand exchange does not result in the introduction of DNA crossings.

Both the Dna replication and homologous recombination mechanisms appear to be conserved in all organisms. S. West (South Mimms, Uk) described the steps that atomic number 82 to initiation of homologous recombination, the formation of recombination intermediates through the activities of RecA and its eukaryotic Rad51 homologues, and the ways in which these recombination intermediates can be processed. The four-style HJ is a central intermediate in such reactions and, in bacteria, the 3 Ruv enzymes grade a 'resolvosome' that acts to move the position of the HJ branch point by branch migration and to resolve the HJ intermediates to recombinant products. A comparable activity in eukaryotic nuclei has been elusive. Nevertheless, West presented evidence for an activity from calf testis that has been partially purified and can catalyze both branch migration and HJ resolution.

Homologous recombination is usually initiated by the formation of double-strand breaks. In eukaryotes, such breaks can also be substrates for a non-homologous end joining (NHEJ) reaction, particularly in cells that are not undergoing Deoxyribonucleic acid replication or meiosis. NHEJ is also used to complete the Dna rearrangement reactions that generate productive antibody and T-cell receptor genes. Indeed these rearrangements link double-strand intermission repair with the procedure of genetic transposition (Grand. Gellert, Bethesda, Md). The proteins RAG1 and RAG2 initiate the 5(D)J recombination reaction in cells of the immune system past making double-strand breaks at the border of recombination signal sequences and the neighbouring coding DNA. Hairpins form at the coding DNA ends (every bit in some other transposition reactions) and, in role, are responsible for introducing genetic diverseness. In normal recombination, the coding ends are rejoined to make novel antibody and T-prison cell receptor genes. Gellert showed that the RAG proteins are also capable of transposing recombination bespeak sequences to new sites, in reactions that resemble those mediated by retroviruses and other characterized transposable elements. Such reactions may crusade translocations that lead to lymphatic tumours.

Although biochemists are used to studying purified DNA and its processing with purified enzymes, Thou. Muniyappa (Bangalore, Bharat), reminded u.s.a. that Deoxyribonucleic acid is simply one component of the circuitous chromosomes that are found within the jail cell. During homologous recombination in meiosis, specialized synaptonemal complexes form between homologous chromosomes. These are highly ordered complex structures that incorporate key recombination proteins. One such meiotic protein is Hop1 which, in in vitro experiments, appeared to collaborate robustly with M quadruplexes commonly found at the ends of chromosomes.

Dna is constantly subjected to harm, both from intracellular process such as oxidation, deamination and base loss, and from exogenous sources like radiation and Dna dissentious chemicals. Furthermore, the processes of replication and recombination themselves tin can pb to mismatches in DNA. Several presentations addressed the mechanism of action of glycosylases that remove abnormal or damaged bases and mismatch repair proteins (1000. Morikawa, Osaka, Japan; A. Bhagwat, Detroit, MI; U. Varshney, Bangalore, India; B. Connolly, Newcastle, UK). Whereas nearly of these speakers discussed the structure and detailed biochemical properties of individual proteins, I. Matic (Paris, France) addressed the dependence of the variable rate of bacterial evolution on the activeness and horizontal transfer of mismatch repair genes. Lack of action of mismatch repair genes leads to high spontaneous mutation rates and loftier homologous recombination frequencies betwixt homologous genes. During adaptation, strains defective mismatch repair are favoured, whereas they are disfavoured once adaptation has been achieved. Analysis of mismatch repair genes amongst many populations shows them frequently to exist composed of DNA sequences from different phylogenetic lineages, an ascertainment consistent with horizontal transfer and frequent loss and proceeds. I. Kobayashi (Tokyo, Japan) also addressed the question of bacterial development by describing how RM systems, which can be considered as selfish mobile Deoxyribonucleic acid elements, may have spread throughout the bacterial kingdom.

Synopsis

The symposium showed that it is the combination of methods (genetics, genomics, enzymology and construction analysis) that has led to major breakthroughs in our agreement of DNA enzymes. Furthermore, a recurring theme was the unexpected similarity amidst many of these enzymes, not only in terms of structure (Figure ​ 3) but also in mechanistic details regarding target site location, Dna recognition and catalysis (Figure ​ 4). These aspects stimulated many fruitful discussions between scientists coming from different fields and made the meeting very successful. In this context it is a pleasure to notation that one of the highlights of the conference was the quality of the posters from graduate students and postal service-doctoral workers and the enthusiasm with which they were defended.

​

The IBUMB symposium on 'Deoxyribonucleic acid enzymes: structures and mechanisms' was held in Bangalore, Republic of india, Dec 1–3, 2000 (organized by V. Nagaraja and D.North. Rao).

The participants of the IUBMB meeting on 'DNA enzymes: structures and mechanisms', gathering for a group photo in front end of the statue of J.N. Tata, the founder of the Indian Institute of Science (photograph provided by the organizers).

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