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Plant DNA Topoisomerases: Structure, Function, and Cellular Roles in Plant Development

Posted on: Wednesday, 14 July 2004, 06:00 CDT

Regulation of the topological state of DNA is critical for cellular viability. DNA topoisomerases alter DNA topology and participate in nearly all events related to DNA metabolism such as replication, transcription, recombination, and chromosome segregation. Despite significant progress in our understanding of the structure, mechanism, and the role of topoisomerases in diverse cellular processes in bacteria, yeast, and animal systems, the roles of plant topoisomerases are poorly understood. In recent years expression studies, characterization of recombinant topoisomerases, and gene knockout studies have highlighted the importance of topoisomerases in plant DNA replication, mitosis, cell proliferation, organelle division, meiotic recombination, morphogenesis, and plant development. We have focused this review specifically on plant topoisomerases, with an emphasis on their biological role in plant development.

Keywords gyrase, replication, cell proliferation, endoreduplication, DNA relaxation, recombination

Table of Contents

I. INTRODUCTION

The genetic information in DNA is in the form of a linear array of nucleotides, but it is the three-dimensional structure of the double helix that regulates most of its cellular functions. The intertwining of the two antiparallel strands of DNA double helix is subjected to a plethora of topological problems in every biological transaction of DNA. A class of enzymes known as topoisomerases resolves the topological demands of DNA (Figure 1). The existence of these topoisomerases has been reported in every living cell. The discovery of DNA topoisomerase I, which specifically relaxes negatively supercoiled DNA, was first reported in Escherichia coli (Wang, 1971) and later identified in mouse cell extracts that relax both positively and negatively supercoiled DNAs (Champoux and Dulbecco, 1972). Another class of enzyme known as DNA topoisomerase II was discovered in E. coli (DNA gyrase), and it is capable of introducing negative supercoils into a relaxed DNA in addition to relaxing supercoiled DNA (Geliert et al., 1976). Later topoisomerase II was identified in humans (Baldi et al., 1980), Drosophila (Hsieh and Brutlag, 1980) and yeast (Liu et al., 1980). Additionally, a reverse gyrase capable of introducing positive supercoils into DNA in an ATP-dependent manner has been characterized from hyperthermophiles (Kikuchi and Asai, 1984; Kozyavkin et al., 1994). Presently many different topoisomerases have been identified in both prokaryotic and eukaryotic systems; their genes have been isolated and the full-length and different domains of the recombinant topoisomerases have been characterized (Wang, 2002). In contrast to bacterial, viral, animal, and yeast systems, where the topoisomerases have been extensively studied, there are relatively few reports on plant topoisomerases. Excellent comprehensive reviews on topoisomerases have been written (Champoux, 2001; Isaacs et al., 1998; Nitiss, 1998; Wang, 1996, 1998, 2002), including a recent one on plant topoisomerase VI (Corbett and Berger, 2003). These reviews do not cover the plant topoisomerases in detail. The purpose of this article is to review recent progress in research on plant topoisomerases. Nevertheless, characteristics of topoisomerases in general have been given to introduce the subject.

FIG. 1. DNA topological interconversions catalyzed by topoisomcrase: (a) introduction or removal of superhelical turns, (b) formation or resolution of catenanes, and (c) interconversion of topological knots and rings. All of these reactions catalyzed by certain class of topoisomerases. However, supercoiling is unique to bacterial gyrase (introduce negative supercoiling in ATPdependent manner), reverse gyrase (introduce positive supercoiling in ATPdependent manner), and pea topoisomerase I (introduce positive supercoiling in ATP-independent manner).

II. CLASSIFICATION OF TOPOISOMERASES

DNA topoisomerases fall into two categories, -type 1 and -type II, on the basis of functional and mechanistic distinctions (Figure 2; Table 1). Type 1 topoisomerases are monomeric proteins, and they change linking number of supercoiled plasmid DNA in steps of one () by transiently cleaving one strand of DNA at a time. They are further divided into two subfamilies, type IA and type IB, on the basis of their mode of action. The members of each family subtype are entirely unrelated in terms of primary amino acid sequence and tertiary structure, but they perform similar functions. Type IA topoisomerases are attached to the 5' phosphate, whereas type IB topoisomerases are attached to 3' phosphate of the nicked DNA strand. Type II topoisomerases are essentially dimers, but in some cases the subunits are split during evolution. The genes encoding the individual polypeptides may fuse together and result in homodimers, as in the case of eukaryotic topoisomerase II, or the genes may further break up and result in hexamer, as in the case of phage topoisomerase II (Champoux, 2001). The type II topoisomerases use a pair of catalytic tyrosines, one in each half of the dimer, to make staggered cuts in both the strands of duplex DNA and changes in the linking number of supercoiled plasmid DNA in steps of two (). They are further divided into two subfamilies, type IIA and type UB, on the basis of structural considerations. Type IIA enzymes are dimeric in nature and leave a 4 bp overhang in cleaved DNA, whereas type HB are tetrameric and leave a 2 bp overhang in cleaved DNA. The type UB subfamily has expanded recently with the discovery of a novel type II, a topoisomerase VI from the hyperthermophilic archaea Sulfolobus shihatae (Bergerat ci al., 1997; Buhler et al., 1998), which is made up of A and B subunits. More recently, the type UB subfamily has been identified in arabidopsis (Hartung and Puchta, 2000,2001 ).

FIG. 2. Formation of a covalent intermediate between DNA and a topoisomerase. Nucleophilic attack of active site tyrosine of topoisomerase on phosphodiester bond of DNA backbone leads to the breakage of the DNA strand and the simultaneous formation of a phosphodiester bond between the enzyme and the DNA. In type IA, IIA, and HB topoisomerases, the tyrosyl group attack from the opposite side of a 3'-oxygen, and the enzyme tyrosyl becomes linked to a 5'- phosphoryl group of DNA in the covalent intermediate (a, c, and d) whereas in case of type IB topoisomerases (b), the enzyme tyrosyl group attacks from the opposite site of a 5'-oxygen and becomes linked to a DNA 3'-phosphoryl group in the covalent intermediate. The type IA and IB topoisomerase nicks the single stranded DNA of duplex (a and b). Type IIA topoisomerase make staggered cut to the duplex DNA and leave 4 bp overhang (c), whereas type UB topoisomerases leave a 2 bp overhang.

TABLE 1

General properties of topoisomerases

III. CRYSTAL STRUCTURE AND MECHANISM OF TOPOISOMERASES

The domain organization of lopoisomerases is depicted in Figure 3. Until recently, information regarding high-resolution structural analysis was not available due to the large size and structural complexity of topoisomerases, hindering their comprehensive biophysical understanding. But now the threedimensional structures of eleven DNA topoisomerase fragments have been solved using X-ray crystallography and nuclear magnetic resonance (see Champoux, 2001). This recent structural information about fully active topoisomerases has been of great help to illuminate the mechanistic action of these enzymes. The mode of action of all topoisomerases is basically the same: a tyrosine residue in the active site of topoisomerase temporarily forming a phosphodiester bond by transesterification in order to generate strand scission in the DNA backbone (Figure 2). After strand-passage through the broken strand, the DNA is religated by exact reversal of the transesterification reaction to transfer the phosphodiester bond from tyrosine to the DNA backbone (Wang, 1996; Watt and Hickson, 1994).

A. Type IA

The main body of type IA topoisomerase is folded together to form a base and a lid having a central hole to accommodate single- or double-stranded DNA (Figure 4a). The active-site tyrosine is buried at the interface of the two domains, and the central hole and active site become accessible only after a substantial conformational change in the molecule, i.e., by movement of lid and base. This type of structure predicts the opening of domains to allow exposure of the active site and to allow entry and exit of DNA, leading to enzyme-bridging-strand passage (Figure 4a) by nicking a DNA strand. A gap is opened between the broken strand to allow passage of the second strand, followed by religation of the nicked strand (Lima et al, 1994; Yu et al., 1995; Mondragon andDiGate, 1999; Champoux, 2001). Recently, the crystal structure of reverse gyrase in the presence and absence of the nucleotide cofactor was reported (Rodriguez and Stock, 2002).

B. Type IB

The structure of the C-terminus domain of vaccinia virus and human topoisomerase I in complex with DNA has been solved (Chenge et al., 1998; Redinbo el at, 1998; Sharma et al., 1994). Human topoisomerase I (type IB) contains four major domains (Figure 3b): a 24-kDa N-terminal domain that has a highly charged NLS and protein- protein interaction domain, a 54-kDa conserved core domain involved in interaction withDNA, a 7-kDa positively charged linker domain that is not required for activity but contributes to processivity of the enzyme, and a 6.3-kDa C-terminal domain that is highly conserved and contains an active-site tyrosine. This enzyme is a bilobed protein that acts like a clamp, completely surrounding its substrate like a nut fitting around a bolt (Figure 4b).

C. Type UA

The complete structure of an intact functional type IIA topoisomerase is lacking. The N-terminal domain of E. coli DNA gyrase B (GyrB) (residues 2 to 398) complexee! with adenosine 5'- [[beta],[gamma]-imidio] triphosphate (ADPNP) was the first model to elucidate structural information for the ATP-binding site of type HA enzymes (Brino et al, 2000; Wigley et al, 1991). Structural insight into DNA binding and cleavage core of type II enzyme became available with information on the crystal structure of the S. cervisiae protein (residues 410 to 1202) (Berger et al., 1996; Pass et al., 1999), by transmission electron microscopy of its truncated derivatives (Benedetti et al., 1997), and from scanning transmission electron microscopy of human topoisomerase II (Schultz et al., 1996). In addition, crystal structure information is available for the N-terminal domain of the gyrase A-subunit (Morais Cabrai et al., 1997). Analysis of these available structures, however, provides a near-complete view of how these amazing proteins are assembled and function. The fulllength enzyme contains a tripartite structure, in which a middle globular-core DNA-binding domain is connected to a pair of small spherical lobes containing an ATPase domain towards the N-terminus region and a small clamplike domain towards the C- terminus region of topoisomerase II. The overall structure is heart- shaped with a large central hole. The molecular model that emerged from these studies (Figure 5a) predicts that the enzyme has an ATP- modulated clamp with two sets of jaws at opposite ends connected by multiple joints.

FIG. 3. Domain structure of different subclasses of topoisomerases. A schematic representation of general domain structure of type I and 11 topoisomerase is shown. Domain boundaries are based on amino acid sequence homology, limited proteolysis analysis, and crystal structure of protein, and are drawn approximately to scale. The +++ indicates highly conserved, ++ indicates conserved, and + indicates poorly conserved region, (a) Domain structure of type IA topoisomerase. The highly conserved N- terminus region contains the active tyrosine and cleavage/ religation domain, a conserved Zn (Il)-binding domain, and a poorly conserved basic C-terminus domain dispensable for enzymatic activity, (b) The type IB topoisomerase comprises an N-terminus poorly conserved domain containing targeting signals, a highly conserved core domain responsible for all catalytic activities except the active site tyrosine, which is located to conserved C- terminus region. A poorly conserved linker region dispensable for enzymatic activity is sandwiched between core domain and C-terminus domain, (c) Domain organization of type HA and B topoisomerases. The homodimer eukaryotic type II topoisomerase contains a highly conserved N-terminus ATPase domain, a conserved central cleavage/ religation domain containing active tyrosine, and a poorly conserved C-terminus domain containing nuclear targeting signals, which is dispensable for supercoiled DNA relaxing activity of the enzyme. The hetrotetrameric bacterial gyrase is comprised of a B subunit containing ATPase domain and aligned to N-terminus region of eukaryotic type IIA topoisomerase, and an A subunit containing active tyrosine and aligned to C-terminus region of eukaryotic type IIA topoisomerase. The hetroterameric topoisomerase VI, a type IIB topoisomerase, contains B subunit aligned to N-terminus region of eukaryotic type UA is topoisomerase and is comprised of ATPase domain, whereas the A subunit contains active tyrosine and is responsible for DNA binding, cleavage activity, and is homologous to Spol 1 genes found in yeast and other systems.

D. Type UB

The less extensively studied type UB enzymes share several common mechanistic features with the type UA enzymes (Bergerat et al., 1997), but there are distinct structural differences between the two subfamilies (Berger et al., 1998; Figure 3c). Type UB DNA topoisomerases are widely distributed (Wang, 2002; Corbett and Berger, 2003). The type UB enzyme is composed of two subunits A (homologous to yeast Spo 11) and B that forms a heterotetramer(A2B2) (Bergerat etal., 1994; Buhler et al., 1998; Figure 3c). The crystal structure of Methanococcus jannaschii DNA topoisomerase VIA subunit fragment from 69 to the end of the protein at residue 389 (Nichols et ai, 1999) confirms the designation of these subunits and provides a number of key insights into how this novel type UB enzyme functions (Figure 5b). Each protomer of the dimeric structure can be divided into two clearly defined domains that overall are distinct from the three-dimensional structures of the type IIA enzymes.

FIG. 4. Molecular models for the passage of one DNA strand through another by type I lopoisomerase. (a) The one strand of duplex DNA is cleaved by a type IA enzyme. The 5' end of broken DNA is attached covalently to the active tyrosine in the lid, and the 3' end is noncovalently bound to the base of the enzyme. Lifting the lid away from the base separates the two ends of broken DNA strand for the passage of another DNA strand or another duplex in the case of catenation/decatenation, known as enzyme bridge mechanism, (b) The type IB enzyme binds to the duplex DNA and cleaves one strand of DNA. The 3' end of cleaved DNA is covalently bound, whereas the 5' end is loosely bound to the enzyme by ionic interaction, which allows the loosely bound end of DNA to rotate in either direction with respect to the other end of DNA. This DNA-rotation mechanism allows multiple strand-passage events for each strand-breakage and rejoining (see Champoux, 2001).

IV. ENERGETICS OF TOPOISOMERASE ACTION

ATP hydrolysis is normally required to drive reactions that are energetically unfavorable. Type I topoisomerases relax supercoiled DNA in the absence of ATP. Supercoiled DNA has a higher free energy than relaxed DNA, and this energy is stored in the form of torsional stress. Gyrase can introduce negative supercoils into DNA, an energetically unfavorable reaction that is coupled to ATP hydrolysis (Bates and Maxwell, 1989; Cullis etal., 1992; Maxwell and Geliert, 1986; Westerhoff et al., 1988). In the absence of ATP, gyrase can catalyze the relaxation of negatively supercoiled DNA, an energetically favorable reaction (Geliert et al., 1977; Sugino et al., 1977). Indeed, in the case of reverse gyrase, which is able to introduce positive supercoils, the requirement of ATP hydrolysis is convincing. Until recently, it was not clear why a eukaryotic type II enzyme performing energetically favorable reactions (relaxation of supercoiled DNA) requires ATP. Now, however, there is strong evidence that ATP hydrolysis is required to shift the equilibrium in the direction of simplification of topology; i.e., to a nonequilibrium distribution of products. Mechanistically, ATP hydrolysis is required for enzyme turnover and strand passage reaction and not for cleavage or rejoining step (Baird et al., 1999). Reddy et al. (1998) reported a topoisomerase I from Pisum sativum that can relax both negative and positive supercoiled DNA in a Mg^sup 2+^- and ATP-independent manner. The striking feature of this enzyme is that in presence of Mg^sup 2+^ it can generate positive supercoiling into relaxed DNA without utilizing ATP. This puzzling finding, where an energetically unfavorable reaction is driven in absence of ATP, is unique among topoisomerases reported so far.

V. PLANT TOPOISOMERASES

The plant topoisomerases have been purified either partially or to apparent homogeneity from wheat germ (Dynan et al, 1981), chloroplasts (Pyke et ai, 1989), and mitochondria (Echeverria et al., 1986, 1991); cauliflower (Fukata and Fukasawa, 1982; Fukata et at., 1986, 1989, 1991); cultured tobacco cells (Heath-Pagliuso et al., 1990; Cole et al., 1992); Pisum sativum root (Chiatante et al., 1991, 1993; Chiatante and Bryant, 1994),shoot(Rudenko, 1992a, 1992b;Levi etal., 1994), and chloroplasts (Lam and Chua 1987;Neilsen and Tewari 1988; Mukherjee et ai, 1994); spinach (Siedlecki et al., 1983), carrot (Carbonerae et al., 1988, 1990, 1995); Brassica oleracea (Kleber et al., 1992a); rice (Agris et ai, 1990; Yoshida et al., 1991); and maize (Carballo et al., 1991). In many of these cases only the presence of a topoisomerase activity or its partial purification has been reported. Predominantly, these enzymes have been purified from whole tissue or partially enriched subcellular organelles. Based on their biochemical properties, such as ATP and divalent ion requirement, and introducing and/or relaxing negative or positive supercoiling and changing the linking number, they are classified as type I or type II topoisomerases and also assigned as eukaryotic or prokaryotic.

FIG. 5. (a) Proposed two-gate mechanism for DNA transport by type IIA topoisomerases. Topoisomerase in open clamp form binds to a DNA duplex (G segment; step 1) and a conformational change to the A' domains occurs (step 2). Another duplex DNA (T segment) and ATP then bind the protein (steps 3). ATP binding leads to the dimerization of the ATPase domains, cleavage of the G segment, and the transport of the T segment through the double-stranded DNA break in G segment (steps 3, 4), then the G segment is religated (step 5), and the T segment escapes through the dimer interface in the A' region (step 6). (b) The proposed molecular model for DNA transport by type IIB topoisomerase. Binding of duplex DNA to the enzyme (step 1) leads to conformational change in the enzyme. Following this the ATP binds and B subunit d\imerize and then the second duplex is trapped by enzyme (step 2). The first duplex is then transiently cleaved and separated to form an opening (step 3), through which the second duplex is transported and cleaved DNA is religated (step 4). Following this the enzyme is recycled for next reaction.

The total genetic material of the plant cell is distributed in three different compartments-nucleus, chloroplast, and mitochondria- and each compartment has its own replication, transcription, and translation machinery to express its genetic material. One would expect the existence of at least three different sets of topoisomerases in the plant system to address the topological problems of the DNA. A number of topoisomerases are expected in the plant cell, and if they are purified from the whole tissue extracts or from the enriched subcellular fractions (it is difficult to purify the subcellular fractions without crosscontamination because of the harsh treatment required to break the rigid cell wall), it becomes difficult to distinguish them from one another based on the molecular size because of the possible proteolysis during purification. These proteolysed fragments are also enzymatically active in terms of DNA relaxation. As the mode of action of all the topoisomerases is basically the same in terms of supercoiled DNA relaxation, it is difficult to establish the identity based on molecular size of the topoisomerases and also subcellular localization if purified from the total plant extracts.

VI. GENES ENCODING PLANT TOPOISOMERASES

Recently, a number of cDNA clones encoding both type I and type II topoisomerases were isolated and characterized from different plant species including arabidopsis (Kieber et al., 1992b; Xie and Lam, 1994a, 1994b), carrot (Balestrazzi et al., 1996, 2000), pea (Reddy et al., 1998, 1999), and tobacco (Mudgil et al., 2002; Singh et al., 2003).

TABLE 2

Topoisomerase genes isolated from plants

The availability of the nucleotide sequence information of the genes encoding different plant topoisomerases has helped in establishing the identity of the encoded topoisomerase in terms of estimated molecular mass and assigning it to different subclasses. The predicted primary amino acid sequences of the plant type I topoisomerases reported so far (Table 2) are similar and contain 800 to 900 amino acid residues with an estimated molecular mass of 80 to 100 kDa. The multiple sequence alignment of the deduced amino acid sequences showed extensive homology among the plant type I topoisomerases. More than 70% of the amino acids were identical in the C-terminus half of the protein with very few insertions or deletions. The colinearity or the sequence is less stringent towards the N-terminus portion of the protein. All the conserved motifs present in other eukaryotic type I topoisomerases (Caron and Wang, 1994) are also present in the same spatial arrangement in the plant type I topoisomerases, suggesting an evolutionary conservation of the overall structure among the eukaryotic type I gene family. Recently, the genomic clones for topoisomerase I have been isolated from tobacco. The sequence analysis of the topoisomerase I genomic clone revealed the existence of 8 exons and 7 introns within the coding region and one intron in the 3' UTR region (Mudgil et al., 2002). It was found that in arabidopsis both of the genes for eukaryotic type I topoisomerase (on chromosome number 5, BAB08547 and BAB08548) have 14 small introns distributed throughout the ORF except in the last part of the conserved core and C-terminus region. Besides variable gene structure, the catalytic domains of topoisomerase I from both arabidopsis and tobacco share similar features and are devoid of introns.

The predicted primary structure of all eukaryotic type II topoisomerases characterized thus far are similar and contain 1429 to 1530 amino acid residues (Wang, 2002) with an estimated molecular mass of 160 to 170 kDa. The eukaryotic topoisomerase II consists of a N-terminus ATPase domain, a central catalytic DNA breakage- rejoining domain, and a C-terminus domain that is required for nuclear localization. All these structural domains are well conserved in the primary amino acid sequences of plant topoisomerase II reported thus far (Table 2). In addition, the seven highly conserved regions found in all known type II topoisomerases (Austin and Marsh, 1998) are also present in the plant topoisomerase II amino acid sequences in a similar spatial arrangement, which could indicate a common origin for these proteins. The bacterial topoisomerase II (DNA gyrase) comprises two subunits, GyrA and GyrB, which bear a striking sequence similarity to the plant topoisomerase II DNA-binding domain and ATPase domain, respectively. It has therefore been suggested that the eukaryotic topoisomerase II probably evolved by the fusion of two gyrase subunits into a single polypeptide (Lynn et al., 1986; Trigueros and Roca, 2002). The multiple sequence alignment of the deduced amino acid sequences shows extensive homology among the plant type II topoisomerases. More than 70% of the amino acids are identical in the N-terminus half, and the colinearity of the sequence is less stringent towards the C-terminus portion of the protein.

Recently the tobacco TopII gene sequence was published (Acc# AY169239). It contains ISexonsand 17 introns, with the majority of the introns of the phase 0 type (Singh et al., 2003), which may result in many splice variants, as reported in the animal system (Petruti-Mot and Earnshaw, 2000), and play a role in developmental processes. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG (our unpublished data) shown by other plant introns (Hebsgaard et al., 1996). Based on the distribution and location of introns of tobacco topoisomerase II compared to that of arabidopsis with respect to their aligned amino acid sequences, it is evident that the intron/exon boundaries are highly conserved between the two genes. However, the length and number of introns in tobacco is different from that in arabidopsis TopII, which contain 20 exons and 19 introns. The high degree of conservation of the intron position and phasing in tobacco TopII and arabidopsis TopII suggests that the two genes could have Originated from a common ancestral gene that might have evolved after a fusion of two genes, analogous to bacterial gyrB and gyrA encoding for the N-terminus and C-terminus domains of the topoisomerase II necessary for its catalytic function.

For the first time outside of the archaebacterial kingdom a homologue of the subunit B of topoisomerase VI has been reported from Arabidopsis thaliana and found to interact with AtSpo11-2 and AtSpo11-3 to form a topoisomerase-VI-like activity (Hartung and Puchta, 2001), suggesting that AtSpo11-2 and AtSpo11-3 are similar to the A subunit of topoisomerase VI. Unlike other eukaryotic systems where only one Spo11gene was reported, in A. thaliana there are three Spoil like genes: AtSpo11-1, AtSpo11-2, and AtSpo11-3 (Hartung and Puchta, 2000, 2001). The AtSpo11-1 gene contains 14 exons, 14 introns in ORF, and one intron in 3' UTR region; the AtSpo11-2 gene contains 11 exons, 11 introns in ORF, and one intron in 3' UTR region (Hartung and Puchta, 2000). The Spo11-3 gene is interrupted by a single intron. Arabidopsis topoisomerase VIB gene contains 18 introns and 19 exons (Hartung and Puchta, 2001).

VII. ORGANELLAR TOPOISOMERASES

Several plastid genomes of higher plants have been completely sequenced (Hiratsuka et al., 1989; Shinozaki and Sugiura, 1986), but no gene encoding for topoisomerases could be identified in the plastid genome. Thus, it is likely that the plastid-specific topoisomerase activities identified are nuclearencoded products targeted to the chloroplast. The cDNA clones for putative DNA gyrase A and DNA gyrase B from pea were isolated as partial overlapping clones by PCR amplification. The deduced amino acids show a significant homology with the corresponding E. coli gyrase A and B. Pea gyrases contain an additional stretch of amino acids at their N- terminal that seems to be a signal for chloroplast targeting (B. Singh et al., unpublished data). Analysis of the A. thaliana genome database reveals the presence of a putative gyrase A (locus: At3g10690) and three gyrase B genes (loci: At3g10270, At5g04130, At5g04110) {initiative, 2000 #3823}. The presence of organellar- targeting sequences suggest that the GyrA protein is targeted both to the mitochondrion and the chloroplast, while one GyrB protein is targeted to the mitochondrion, one to the chloroplast, and one is not targeted. A. thaliana seedlings and cells are sensitive to gyrase-specific drugs, and DNA supercoiling activity can be detected in both mitochondria and chloroplast extracts. The A. thaliana genes have been cloned and expressed in E. coli and shown to possess supercoiling activity (M. K. Wall, L. A. Mitchcnall, and A. Maxwell, manuscript in preparation). A putative type IA found in the arabidopsis genome database on chromosome 4 (Accession # 18417755). Comparison of the deduced amino acid sequence with prokaryotic topoisomerase showed that arabidopsis type IA topoisomerase contains an additional stretch of sequence to its N-terminal region that seems to be the chloroplast signal peptide. The genes encoding the plastid-specific topoisomerases are evolutionarily transferred to the nucleus but retain the prokaryotic nature of their encoded proteins in terms of the primary amino acid sequence homology with prokaryotic topoisomerases. However, the genes encoding these proteins gained many eukaryotic properties, like the presence of introns and polyadenylation. There are 24 introns in gyr A (NM_111905), 19 introns in gyr B (NMJ 11862), and 20 introns in type IA topoisomerase (NM_119270).

Biochemical studies of purified mitochondria from various sources indicate t\hey contain type IB topoisomerase with properties different from those of nuclear topoisomerase I. Several animal and plant mitochondrial genomes have been sequenced completely, but no ORF has been found to code for the topoisomerase. Thus, it is likely that the topoisomerase activity reported in mitochondria of animals and plants is due to enzymes encoded by the nuclear genome. The genes encoding mitochondrial topoisomerase II have been reported from trypanosoma (Fragoso and Goldenberg, 1992; Strauss and Wang, 1990) and Dictyostelium discoideum (Komori et al., 1997). The presence of a distinctive type IB topoisomerase in mitochondria has received strong support by the identification of a human chromosomal gene (8q24.3) encoding a mitochondria-targeting type IB topoisomerase (Zhang et al., 2001). Recently, it has been demonstrated that the human topoisomerase III[alpha], which was cloned earlier (Hanai et al., 1996), is present in both the nucleus and mitochondria of human cells (Wang et al., 2002). Although topoisomerase activity has been reported from plant mitochondria (Echeverria ei al., 1986, 1991; Meissnere/o/., 1992), genes encoding plant mitochondrial topoisomerases have yet to be discovered.

VIII. BIOLOGICAL ROLES OF PLANT TOPOISOMERASES

A. DNA Replication and Organelle Division

The role of topoisomerases in initiation of the replicating bubble, elongation of replicating DNA chains, and segregation of newly replicated chromosomes has been extensively studied in bacterial and animal systems (Nitiss, 1998; Wang, 2002). The topological problem associated during elongation of replication bubble is depicted in Figure 6a.

A role for plant topoisomerase I in replication was first shown in chloroplasts in vitro (Nielsen and Tewari, 1988). The addition of purified pea chloroplast topoisomerase I to a replication system containing DNA polymerase, DNA-binding proteins, and other proteins involved in replication resulted in a significant stimulation of replication activity on the recombinant supercoiled DNA templates that contained the pea chloroplast DNA replication origins. A similar result was obtained when pea chloroplast topoisomerase I was replaced with E. coli topoisomerase I, but a eukaryotic topoisomerase I from calf thymus did not stimulate replication (Nielsen and Tewari, 1988).

The role of DNA gyrase in plastid replication in cultured cells of Nicotiana tabacum has been investigated by applying low concentrations of nalidixic acid, an inhibitor of DNA gyrase that affects plastid DNA rather than nuclear DNA synthesis. Removal of the drug from the culture medium led to the resumption of DNA synthesis (Heinhorst et al., 1985). Mills et al. (1989) also showed that incorporation of thymidine into DNA of isolated pea chloroplasts was inhibited by nalidixic acid and novobiocin of gyrase. During the development of plastids, the maximal level of gyrase was found at the time of replication of the plastid DNA, and the localization of gyrase by immunolabelling coincides with that of plastid DNA replication (Marrison and Leech, 1992). Later Itoh et al. (1997) studied the effect of nalidixic acid on the replication and division of organelle nucleoids in a synchronous Cyanidioschyzon merolae culture. Most of the chloroplast nucleoids divided unequally. These results suggested that the unequal division of the chloroplast nucleoid may be due to the inhibition of decatenation by gyrase in the presence of nalidixic acid, making the duplicated chloroplast nucleoids physically unable to segregate, as demonstrated in gyrase mutants of E. coli (Steck and Drlica, 1984). Pyke et al. (1989) had shown the accumulation of 96-kDa and 101-kDa polypeptides (comparable to E. coli gyrase A and B) to the maximum levels during chloroplast DNA replication by immunostaining with yeast topoisomerase II antibodies. Topoisomerase I has been shown to be associated with wheat mitochondrial active DNA replication complex (Echeverria et al., 1986, 1991). Inhibition of type II topoisomerase resulted in cuplike abnormal mitochondria in Euglena gracitis (Ebringer er al., 1993). The above-mentioned examples clearly show the active involvement of topoisomerases I and II (gyrase) in plastid DNA replication and partition.

FIG. 6. Topological problems associated with replication, transcription, and recombination. (a) During elongation of replication the DNA turns as it is passes through the immobile replication machinery (circle), resulting into accumulation of positive supercoils ahead of the moving fork. This topological problem can be solved by type IB or type II DNA topoisomerases, but probably not by type IA enzyme. (b) When the transcription machinery (circle) cannot rotate around the helical axis of the DNA template, overwinding or positive supercoiling of the DNA template ahead of transcription complex is accompanied by underwinding or negative supercoiling of the template behind complex. (c) During recombination the topological problem arises as knots and tangles. The topoisomerase helps in resolving the Holliday junctions encountered during recombination. It is thought that type IA topoisomerase is especially involved in the resolution of double Holliday junction.

B. Role in Cell Division and Cell Proliferation

The topoisomerases play an important role in chromosome structure and chromosome condensation during mitosis in mammalian cells (reviewed by Nitiss, 1998).

Both type I and type II topoisomerases play a major role in plant development. Transcript analysis shows that the expression of both type I and type II topoisomerase transcripts correlates with cell proliferation in maize (Carballo et al., 1991), arabidopsis (Xie and Lam, 1994a), carrot (Balestrazzi et al., 1996, 2000), pea (Reddy et al., 1999) and tobacco (Mudgil et al., 2002). Arabidopsis TopVIB and AtSpo11-3 mutants have deficient cell proliferation (Hartung et al., 2002). The localization of topoisomerase II by immunofluorescent staining during different stages of mitotic cell division strongly suggests that topoisomerase II plays a significant role in chromosome condensation and resolution of chromatids during plant cell division (B. N. Singh et al., unpublished data). Similarly, in many other organisms topoisomerase II is the structural component of the mitotic chromosome scaffold and plays a role in chromosome condensation (Earnshaw and Heck, 1985; Earnshaw et al., 1985; Gasser et al., 1986; Melendy and Ray, 1987; Rainwater and Mann, 1991; Roberge et al., 1990), and it also plays an essential role in chromatid segregation (Ishida et al., 1994; Shamu and Murray, 1992).

Recently, Sugimoto-Shirasu et al. (2002) have isolated two arabidopsis dwarf mutants that are defective in the hypo gene, which encodes the B subunit of topoisomerase VI and the rh12 gene, which encodes AtSpo11-3 (the A subunit of topoisomerase VI). They proposed that the topoisomerase VI complex is essential for the decatenation of replicated chromosomes during endocycles and that successive rounds of endoreduplication are required for the full growth of specific cell types.

C. Role in Morphogenesis and Plant Development

The functionally defective plant topoisomerase mutants have not been studied extensively to establish their role in plant development. Takahashi et al. (2002), showed that disruption of an arabidopsis topoisomerase I gene affected phyllotaxis, the serration of leaf margins, the twisting of inflorescences, and the internode lengths between two successive flowers. These morphological abnormalities observed in topoisomerase I mutants of arabidopsis suggest that topoisomerase I may play a critical role in regulating the developmental pattern in plants. A sequence analysis of the arabidopsis genome indicates the existence of two type I topoisomerase genes (Accession # NML124912). The functional silencing of both genes was found to be lethal at young seedling stages, suggesting that topoisomerase I activity is essential in plant development. In drosophila and mouse, the topoisomerase I- defective genotype is lethal (Lee et al., 1993; Morham et al., 1996), whereas in lower eukaryotes like yeast, type I topoisomerase mutants are developmentally defective but not lethal (Thrash et al., 1984; Uemura and Yanagida, 1984). Overexpression of tobacco topoisomerase I lead to early flowering in transgenic tobacco plants (B. N. Singh et al., unpublished data).

Recently, Yin et al. (2002) identified two different mutants of arabidopsis, AtSpo11-3 and AtTop-6B. Both mutants showed identical phenotypes and downregulated the same set of genes that were identified by the microarray analysis. This result suggests that AtSpo11-3 and AtTop-6B gene products interact with each other and constitute a functional topoisomerase-VI-like activity that plays a major role in plant growth and development. The dwarf phenotype observed in AtSpo11-3 and AtTop6B arabidopsis mutants was similar to that of brassinosteroidinsensitive mutants. The AtSpo11-3 and AtTop- 6B mutants downregulated more than 18 brassinosteroid-induced genes that encode for cell-wall-modifying proteins. This provides a possible explanation for the similar dwarf phenotype of those of brassinosteroid-insensitive mutants (Darley et al., 2001; Friedrichsen and Chory, 2001). Recently Hettiarachchi et al. (2003) showed light-mediated regulation of pea topoisomerase II by promoter analysis using reporter gene in transgenic plants.

D. Role in Transcription

Topological problems that are encountered during transcription (Figure 6b) are solved by topoisomerases. Topoisomerases have been shown to be involved actively in the transcription process in animal system. In prokaryotic systems the locally negative supercoiled DNA is required for the initiation of transcription (Liu and Wang, 1987; Merino et al., 1993; Nitiss, 1998; Shaiu and Hseih, 1998; Shykind et al., 1997; Uemura and Yanagida, 1984; Wang, 2002). The data on the involvementof topoisomerases on plant genome transcription is not available. The first report of the role of topoisomerases in chloroplast transcription was shown in Chlamydomonas reinharditi (Thompson and Mosig, 1985) by inhibiting the gyrase activity that results in differential accumulation of various chloroplast transcripts. Later, Lam and Chua (1987) observed similar results in pea chloroplast in in vitro transcription assays. The inhibition of different topoisomerases using appropriate drugs results in alteration of the topological state of the DNA, which in turn affects the activation of different promoters differentially by transcription machinery. Nielsen and Tewari (1988) have shown that the supercoiled templates are essential for the proper initiation of the runoff transcripts in their in vitro assays. It has also been demonstrated that topoisomerase I is involved in both repression and activation of different transcripts (Merino et al., 1993). Hoeberichts et al. (2001) reported that the application of campothecin, an inhibitor of topoisomerase I, leads to upregulation of HSR203, CTU1, and CTU2,and downregulation of the DAD1, CTD1, CTD2, CTD3, CTD4, and CTD5 transcripts in tomato cell culture. The arabidopsis mutants FAS1 and FAS2 show pleiotropic phenotypes, including fasciated stem morphology, serrated leaves, and disturbed phyllotaxis (Leyser and Furner, 1992). The corresponding genes encode two of the three subunits of chromatin assembly factor-1 (Kaya et al., 2001). These data highlight the importance of chromatin remodelling and assembly in the control of plant morphogenesis (Verbsky and Richards, 2001). Furthermore, the arabidopsis topoisomerase Ia mutant resembles the phenotype with the above-mentioned mutants (Takahashi et al., 2002). This gives a strong indication that topoisomerase I may be involved in chromatin assembly and remodelling and hence may play a role in transcriptional regulation.

E. Involvement of Plant Topoisomerases in Mitotic and/or Meiotic DNA Recombination

The role of topoisomerases in recombination has been studied extensively in the last few decades in animal system. Different topoisomerases mutants showed hyperrecombination, instability in rDNA, defect in meiotic recombination, and defect in telomere structure (reviewed in Nitiss, 1998; Wang, 2002). Recently, the role of plant topoisomerase in recombination has received increased attention. Topoisomerases play a major role in legitimate and/or illegitimate DNA recombination. Topological problems that are encountered during recombination are depicted in Figure 6c. Sequence analysis of transgenic integrations in arabidopsis, rice, and tobacco revealed a putative topoisomerase-I-binding site near the junctions, suggesting the role of topoisomerase I in DNA recombination (Kohli et al., 1999; Sawasaki et al., 1998; Shimizu et al., 2001). This junction was followed by a short track of purine- pyrimidine residues (TATATAGA) followed by another similar alternating sequence (ATATCACAT) 50-bp upstream. oSuch sequence motifs are known to adopt a Z-DNA conformation, and these structures are known to influence the recombination (Holliday, 1989). Topoisomerase IIs are known to bind these Z-DNA structures (Spitzner et al., 1990) and resolve the recombination intermediates (Puchta and Meyer, 1994; Papp et al., 1996). The topoisomerase II cleavage sites and the A-T-rich purine-pyrimidine tracks are known to be cis elements of scaffold-attachment regions (Slatter et al., 1991). The insertion of scaffold-attachment regions at 5' and 3' sites of the nptII transformation cassette increases the transformation efficiency five- to ten-fold in cultured tobacco cells (Shimizu et al., 2001). Scaffold-attachment region-based transformation vectors have been shown to improve transformation frequency in plants (Buising and Benbow, 1994; Galliano et al., 1995; Meyer et al., 1988) and animals (Gutierrez-Adan and Pintado, 2000; Menck et al., 1998). Evidence from mammalian systems indicates that chromosomal rearrangements, including large deletions, are associated with SARs with a possible involvement of topoisomerase II (Pomykala et al., 1994; Sperry et al., 1989).

Genetic recombinations occur during meiosis, and topoisomerases are known to be involved in legitimate and/or illegitimate DNA recombination. However, there is no experimental data showing the involvement of topoisomerases in plant meiotic recombinations. Recently, Grelon et al. (2001) have shown that disruption of AtSpo11- 1, a homologue of topoisomerase VI A subunit, induced perturbation of male and female meiosis, with the formation of very few bivalents at the end of prophase I. The drastic decrease of meiotic recombination observed in arabidopsis Spo 11-1 mutant indicates an absence of crossing over, a prerequisite to any chiasmata and bivalent stabilization similar to Spot I mutant of yeast, drosophila, and C. elegans (Cha et al., 2000; Celerin et al., 2000; Dernburg et al., 1998; Giroux et al., 1989; McKim and Hayashi- Hagihara 1998). The Spoil encoded protein, involved in the induction of double-stranded breaks in yeast, is necessary for crossovers and recombination (Paques and Haber, 1999; Smith and Nicolas, 1998).

IX. REGULATION OF PLANT TOPOISOMERASES

Topoisomerases are regulated at transcriptional, posttranscriptional, and posttranslational levels, and via protein- protein interaction.

A. Cell Cycle Stage-Specific Regulation

Studies in yeast have shown that TopII is a cell-cycleregulated gene. It has been found that TopII is indispensable at the stage where chromosomal segregation and decatenation is required (Uemura and Yanagida, 1984; Holm, 1994; Holm et al., 1985,1989; Uemura et al., 1987). It is already established by extensive studies in various eukaryotic systems that expression of TopII[alpha]-specific mRNA is highest in proliferating tissues, while TopII[beta] mRNA levels do not correlate with proliferation state. Topi, on the other hand, is present in many quiescent cell types (Duguet et al., 1983; Heck and Earnshaw, 1986). In HeLa cells, TopII a mRNA levels rise ten-fold in late S and G2/M phase, compared with the levels in G1 phase, and then decline rapidly following mitosis. In contrast, TopII[beta] mRNA levels show a twofold increase during progression through the cell cycle (Isaacs etal., 1998).

The only example of this kind of study is on synchronized tobacco cell cultures, where both Topi and Topll transcripts were found to be present at a basal level in all stages of the cell cycle. However, the expression level increased from G1 onwards, attaining a maximum during S phase for Topi (Mudgil et al., 2002). This observation agrees with recent data suggesting that the Topl[beta] gene promoter, whose activity is low in carrot protoplasts arrested by starvation in G^sub 0^/G^sub 1^, is activated when protoplasts are induced to re-enter the cell cycle during the G1/S phase (Balestrazzi et al., 2003). The expression pattern of TopII transcript increases approximately three-fold during S-phase and eight-fold during the G2/M phase (B. N. Singh et al., unpublished data). The increased expression level of topoisomerase Il observed during S and G2/M phases is consistent with the argument that topoisomerase II is required during DNA replication, and chromosome condensation and segregation are required during mitosis. Similar expression patterns of topoisomerase II were observed in synchronized HeLa cells (Goswami et al., 1996). Coupling the topoisomerase II transcript level to cell cycle pattern may be controlled at the level of transcription, posttranscriptionally or both. The transcription of genes that are involved in early stages of the cell cycle is regulated largely by transcription factor E2F along with retinoblastoma (Rb) protein. This transcription factor binds to the E2F-binding site in the promoter region and regulates the expression of the transcript. The sequence analysis of the tobacco topoisomerase II promoter region showed the presence of four E2F-binding sites, suggesting that the cell cycle coupled accumulation of topoisomerase II transcript may be controlled at the transcription level. In addition to E2F sites, the tobacco topoisomerase II promoter region also contains ICE elements (CCAAT) that are thought to be involved in cell-cycle-regulated expression of topoisomerase II in human and Chinese hamster cells (Mirski and Cole, 1995). However, the selected accumulation of topoisomerase II transcripts during different stages of cell cycle may be due to the regulation of mRNA stability. Goswami et al. (1996) have shown that the cell-cycle-coupled accumulation of mammalian Toplla mRNA levels is regulated by changes in mRNA stability. During the G2/M phase of cell cycle, this stability was regulated by 3' UTR via interaction with redox-sensitive protein complexes that bind to the AUUUUUA motif present in the 3'UTR of TopII[alpha] gene (Goswami et al., 2000). The sequence analysis of the 3' UTR region of tobacco topoisomerase II also shows the presence of AUUUUUA motif (B. N. Singh et al., unpublished data), suggesting the posttranscriptional regulation of the selected accumulation of topoisomerase II transcript during G2/M phase of the cell cycle.

B. Posttranslational Regulation

Eukaryotic topoisomerases undergo posttranslational modifications by phosphorylation in a number of different organisms. It has been postulated that this may regulate the activity of the enzyme, its interaction with other nuclear factors, or its subcellular localization. Phosphorylation of topoisomerase by CKII and PKC and other kinases such as Ca^sup 2+^/calmodulin-dependent protein kinase, p34cdc2 kinase, ERK1, ERK2, and several MAP kinases in various systems has been reported (reviewed by Wang, 1996; Isaacs et al., 1998).

In vivo both Topo I and II exist as phosphoproteins. Although extensive information is available from animal systems, little is known ab\out how the phosphorylation of topoisomerases in plants can modulate its catalytic activity. Alkaline phosphatase treatment of nuclear extracts abolished topoisomerase II activity in Zea mays (Carballo et al., 1991). The purified topoisomerase I from cultured tobacco cells were marginally affected by phosphorylation or dephosphorylation with regard to DNA relaxation activity (Cole et al., 1992). A 45-kDa putative topoisomerase I from pea root meristem was shown to be phosphorylated In vitro (Chiatante and Bryant, 1994). The deduced primary amino acid sequence analysis showed a number of putative phosphorylation, myristoylation, and glycosylation sites. However, the recombinant plant topoisomerases expressed in E. coli are enzymatically active in terms of supercoiled DNA relaxation without any eukaryotic posttranslational modifications (Reddy et al., 1998; Mudgil et al., 2002). The recombinant pea topoisomerase I activity was found to be stimulated after phosphorylated by Protein Kinase C and casein Kinase II (Tuteja et al., 2003), whereas the tobacco topoisomerase I was phosphorylated by Protein Kinase C but not by casein Kinase II. The phosphorylation, however, did not affect the detectable change in the DNA relaxation activity of tobacco recombinant topoisomerases (Mudgil et al., 2002).

X. PROTEIN-PROTEIN INTERACTION

Topoisomerases are involved in every biological transaction of DNA and are expected to interact with other proteins involved in DNA replication, transcription recombination, and segregation. However, not much data are available on the interaction of plant topoisomerases with other proteins. Topoisomerase I has been reported to interact with PKC, CKII, histone H1, HMG, nucleolin, RNA pol II, p14 (reviewed in Wang, 1996). Yeast topoisomerase II has been shown to interact with casein kinase II, sgs 1, and Pat1p, whereas mammalian topoisomerase II interacts with CKII, CREE, ATF2, c-Jun, 14-3-3[epsilon], retinoblastoma protein, ERKl, ERK2, CAD nuclease, P53, and CD3[epsilon]. Drosophila topoisomerase II has been reported to interact with barren gene product. Human TopII[alpha] interacts with [beta] (reviewed in Bakshi et al., 2001). Interaction of topoisomerase II with PCNA has been shown in chicken (Niimi et al., 2001).

Recently Pham et al. (2000) reported a DNA helicase, which interacts physically with pea topoisomerase I as assayed by immunoprecipitation studies, showed stimulation of DNA relaxation activity by topoisomerase I in the presence of DNA helicase. The stimulation of pea topoisomerase I activity by DNA helicase could be due to local over winding (supercoiling) of DNA template by unzipping the two strands and to the likelihood that helicase- induced DNA supercoiling may generate a preferred substrate for topoisomerase I. These considerations led to the suggestion that both DNA helicase and topoisomerases are required to provide the swivel mechanism for DNA replication. Similar results were observed where host-encoded rep DNA helicase and the phage-encoded, sequence- specific topoisomerase I were found to interact for strand separation of the phage DNA during replication and where eukaryotic topoisomerase I and T antigen (helicase) interact with each other for unwinding the DNA strand at SV40 replication fork (Scott and Kornberg, 1978; Duguet et al., 1983). Using a two-hybrid strategy, Watt and coworkers (1995) found a protein SGS-1, a putative helicase (RecQ homologue) whose mutation was previously described as a top3 suppressor (Wallis et al., 1989) that interacts with the C-terminal domain of topoisomerase 11 in yeast, suggesting a physical as well as functional interaction between helicase and topoisomerase II.

Van-Hop et al. (1999) observed that pea PCNA was competent to form a complex with pea topoisomerase I. The in vivo significance of this interaction remains to be ascertained, particularly in view of the promiscuity of PCNA in forming complexes with a host of other cellular factors. It is apparent that the complex of PCNA and topoisomerase I would keep topoisomerase I in an inactive state. The complex form might offer a storage mechanism for the topoisomerase I activity that is utilized whenever necessary by allowing dissociation of the complex. For example, following the complex formation at the prereplicative stage, PCNA may be taken up from the complex as one of the fork-associated proteins during ongoing DNA replication because the free energy of interaction between PCNA, RFC, and polymerase [delta] is expected to be higher than that between PCNA and topoisomerase I (Bouayadi et al., 1997; Yao et al., 1996). In this event, the decoupled topoisomerase I would be able to relieve the tortional stress at the flanking ends of the DNA replication forks to facilitate DNA replication. As soon as the DNA replication fork reaches the terminus, topoisomerase I might pull PCNA out of the stalled replisome and thereby facilitate recycling of the process of further fork formation and DNA synthesis (Yao et al., 1996).

XI. DNA SEQUENCE PREFERENCE OF PLANT TOPOISOMERASE AND TOPOISOMERASE POISON

The topoisomerase-mediated DNA cleavage is not randomly distributed in DNA. Studies on type I enzymes showed that nine nucleotides on the scissile strand and five on the noncleaved strand were essential for strong binding (Capranico and Binaschi, 1998). The DNA cleavage sites by wheat germ and rat liver topoisomerase I were mapped on duplex S V40 DNA (Been et al., 1984). The frequency of breakage varies greatly from one site to another, with 25% of the sites accounting for greater than 80% of the breakage. These strong sites show preference to cleave at (A/T) (G/C) (T/A) T[arrow down], in the scissile strand. The wheat germ topoisomerase I showed preference for T at -2 position and C (and not T) at -3 position. The nature of ambiguities seen in the -4 to -2 position of the consensus sequence suggests that the recognition of this sequence by topoisomerase I may involve interaction with the minor groove of the DNA. There is no nucleotide bias at the +1 position. (Tanizawa et al., 1993). Type II enzymes from a wide variety of sources preferentially cleave at R [arrow down], Y (purine [arrow down] pyrimidine) repeats (Spitzner et al., 1990), although this consensus is not so stringent and there exist species preferences (Burden and Osheroff, 1999; Sander and Hsieh, 1985; Spitzner and Muller, 1988; Yoshinari et al., 1993). It has been shown recently that drosophila topoisomerase II may cleave at a specific nucleotide of a DNA hairpin by using short oligomers (Froelich-Ammon et al., 1994), which indicates that the structure, not the sequence, was the main determinant of the cleavage activity; whether the same holds true in vivo is not clear.

Topoisomerase inhibitors may not change the cleavage sites of a given DNA substrate by the topoisomerase, but each drug may stimulate DNA cleavage at certain sites, suggesting that effective drug interaction depends on the local base sequence. Drug localization in the ternary complex has been shown using a photoactive mAMSA analog: upon light activation, the compound was found to be covalently linked to DNA bases at the +1 and -1 positions only when T4 topoisomerase II was present in the reaction mixture (Freudenreich and Kreuzer, 1994). Analogous results have been obtained in the case of camptothecin and topoisomerase IB (Jaxel et al., 1991; Pommier et al., 1995). The drug does not affect the interaction between the enzyme and DNA; it only affects the topoisomerase I reaction at some steps after the binding reaction (Champoux, 1988). Based on the biochemical and crystallographic information, as receptor for camptothecin has been proposed (Redinbo et al., 1998). The drug interacts with specific enzyme amino acid residues and with the required GC base pair at position +1 of the DNA cleavage site. These receptors are localized at the protein/DNA interface of the enzyme active site and support the stacking model of campothecin (Jaxel et al., 1991). Espinas and Carballo (1993) showed that treatment of maize protoplasts with VM-26, a topoisomerase II inhibitor, leads to the appearance of stable DNA fragments of about 50 kb in all the tissues. This suggested the folding of DNA in higher order chromatin domain structure. Studies using VM-26 in tissue culture cells showed that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection (Udvardy and Schedl, 1991).

XII. CONCLUSIONS AND PERSPECTIVES

Topoisomerases are involved in many functions related to DNA metabolism, replication, and transcription. Their presence and properties have been well studied in bacterial, yeast, and animal systems. Recently, their role in development during early embryogenesis has been shown. The functions of different isoforms of topoisomerase I and II during mitosis, meiotic recombination, replication, growth, and development are also being elucidated. In this background the data currently available on plant systems is limiting and the exact functions and biological roles of topoisomerases are only beginning to be understood. This review has taken into account the earlier reports on the partial characterization of this group of enzymes from plant systems and recent reports on their role in development.

Plants contain topoisomerases in the nucleus, chloroplast, and mitochondria. However, genes encoding the chloroplast and mitochondria enzymes have not been cloned. The organellar topoisomerases must be nuclear encoded, as the plastid genome sequences of several organisms have been completed and no known replication genes have been found to be encoded by the sequences. In fact, subcellular localization of topoisomerases has not been attempted. It is expected that future work will be directed towards understanding the mechanistic and functioning of organellar topoisomerases.

Though some work has been done, detailed \characterization of nuclear topoisomerases is still lacking. This is partly due to the fact that it is only now possible to produce the recombinant protein in large amounts, and hence work can be undertaken for topoisomerase II, topoisomerase VI, and prokaryotic topoisomerase I. For comparisons it may be imperative to find the crystal structure of plant enzymes in future studies.

The recent studies on knockout mutants of topoisomerases in arabidopsis have revealed their role in plant morphogenesis, development, endoreduplication, and meiotic recombination. More work will have to be done in some other plants also using antisense/RNAi technology to find out the role of individual topoisomerases. There are also indications of the regulation of topoisomerase transcripts in response to light, hormones, and stress, and these are also regulated in cell-cycle-specific manners. An in vivo analysis of their regulatory elements would be of interest. It would be worthwhile to find out the role of topo VI in plants in more detail, because usually this enzyme is present only in archaea. Using yeast two-hybrid, coimmunoprecipitation, and other techniques, the association of topoisomerases with other proteins should be also investigated. There are already indications of topoisomerase I interactions with PCNA and helicases. There is evidence from the work on animal systems that topoisomerases are components of larger protein complexes providing proper modelling and assembly of chromatin, which in turn may also be influencing the expression of a large number of genes in response to one stimulus. These are some of the issues that would attract attention in the future.

ACKNOWLEDGEMENTS

We would like to thank Prof. Anthony Maxwell, Dr. Melisa Wall, Prof. Daniela Carbonera, and Mathilde Grelon for critical reading and their comments on the manuscript. We acknowledge Dr. Mintu Desai for help in computer graphics.

Referee: Professor Stanley J. Roux, Institute for Cellular and Molecular Biology, Department of Botany, University of Texas at Austin, Austin, TX-87713, USA

REFERENCES

Austin, C. A. and Marsh, K. L. 1998. Eukaryoiic DNA topoisomerase II beta. Bioessays 20: 215-226.

Agris, P. E, Parks, R., Bowman, L., Guenther, R. H., Kovacs, S. A., and Pelsue, S. 1990. Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies. Exp. Cell Res. 189: 276-279.

Baird, C. L., Harkins, T. T., Morris, S. K., and Lindsley, J. E. 1999. Topoisomerase II drives DNA transport by hydrolyzing one ATP. Proc. Natl. Acad. Sd. USA 96: 13685-13690.

Bakshi, R. P., Galande, S., and Muniyappa, K. 2001. Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase. CnV. Rev. Biochem. Mol. BM. 36: 1-37.

Baldi, M. I., Benedetti, P., Mattoccia, E., and Tocchini- Valentini, G. P. 1980. In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase. Cell 20: 461- 167.

Balestrazzi, A., Chini, A., Bernacchia, G., Bracci, A., Luccarini, G., Cella, R., and Carbonera, D. 2000. Carrot cells contain two topi genes having the coding capacity for two distinct DNA topoisomerases I. J. Exp. Bot. 51: 1979-1990.

Balestrazzi, A., Ressegotti, V., Panzarasa, L., and Carbonera, D. 2003. Isolation and functional analysis of the 5'-flanking region of carrot top1 [beta] gene coding for the [beta] isoform of DNA topoisomerase I. Biochim. Biophys. Acta 1625: 197-202.

Balestrazzi, A., Toscano, I., Bernacchia, G., Luo, M., Otte, S., and Carbonera, D. 1996. Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation. Gene 183: 183-190.

Bates, A. D. and Maxwell, A. 1989. DNA gyrase can supercoil DNA circles as small as 174 base pairs. EMBOJ. 8: 1861-1866.

Been, M. D., Burgess, R. R., and Champoux, J. J. 1984. Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA. Nucleic Adds Res. 12: 3097-3114.

Benedetti, P., Silvestri, A., Fiorani, P., and Wang, J. C. 1997. Study of yeast DNA topoisomerase II and its truncation derivatives by transmission electron microscopy. J. BM. Chem. 272: 12132-12137.

Berger, J. M., Pass, D., Wang, J. C., and Harrison, S. C. 1998. Structural similarities between topoisomerases that cleave one or both DNA strands. Proc. Natl. Acad. Sci. USA 95: 7876-7881.

Berger, J. M., Gamblin, S. J., Harrison, S. C., and Wang, J. C. 1996. Structure and mechanism of DNA topoisomerase II. Nature 379: 225-232.

Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P. C., Nicolas, A., and Forterre, P. 1997. An atypical topoisomerase II from Archaea with implications for meiotic recombination. Nature 386: 414-417.

Bergerat, A., Gadelle, D., and Forterre, P. 1994. Purification of a DNA topoisomerase II from the hyperthennophilic archaeon Sulfolobus shibatae. A thermostable enzyme with both bacterial and eucaryal features. J. BM. Chem. 269: 27663-27669.

Bouayadi, K., van der Leer-van Hoffen, A., Balajee, A. S., Natarajan, A. T., van Zeeland, A. A., and Mullenders, L. H. 1997. Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin. Nucleic Adds Rex. 25: 1056-1063.

Brino, E., Urzhumtsev, A., Mousli, M., Bronner, C., Mitschler, A., Oudet, P., and Moras, D. 2000. Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center. J. Dial. Chem. 275: 9468-9475.

Buhler, C., Gadelle, D., Forterre, P., Wang, J. C., and Bergerat, A. 1998. Reconstitution of DNA topoisomerase V1 of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli. Nucleic Adds Res. 26:5157-5162.

Buising, C. M. and Benbow, R. M. 1994. Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence. Mol. Gen. Genet

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