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DNA polymerases from plant cells
Mutation Research, 181 (1987) 81-91 Elsevier
81
MTR04379
D N A polymerases from plant cells * Simon Litvak and Michel Castroviejo Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1, rue Camille Saint Sa~ns, 33077 Bordeaux Cedex (France)
(Received 5 January 1987) (Accepted 11 March 1987)
Keywords: DNA polymerases; Plant cells; DNA primase; Topoisomerase; Helicase; Ribonuclease; Ligase; DNA viruses.
The replication and repair of DNA involve the concerted activity of several protein factors and enzymes. These include DNA polymerases, proteins which are associated to the DNA polymerases, DNA primase, topoisomerase, helicase, DNA-binding proteins, ribonuclease and ligase (for a review see Kornberg, 1980). By far the most studied of these proteins are DNA polymerases which are found in all the cellular compartments where DNA must be duplicated. DNA polymerases catalyse the incorporation of deoxyribonucleoside monophosphates (dNMP) in a 5'-3' direction using the parental strands as templates. These enzymes use deoxynucleoside 5'-triphosphates as substrate; pyrophosphate is eliminated in the reaction and a phosphodiester bond is formed between the remaining phosphate of the dNMP and the 3'-OH group of the previously incorporated precursor. The breakage of the alpha-beta phosphate linkage of the deoxyribonucleoside triphosphate provides the energy for the reaction. The sequence of the daughter strands is determined by the insertion of the complementary base following the classical A-T, G - C rule. No DNA polymerase is able to initiate DNA synthesis on a single-stranded DNA in the ab-
* This article is dedicated to Professor L.F. Leloir (Buenos Aires, Argentina) on the occasion of his 80th birthday. Correspondence: Dr. S. Litvak, Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1, rue Camille Saint SaSns, 33077 Bordeaux Cedex (France).
sence of a primer which provides a free 3'-OH able to form the phosphodiester bond with the oncoming precursor nucleoside. RNA primers for DNA initiation are synthesized in the lagging strand of the replication fork by DNA primase (Kornberg, 1980). Prokaryotic organisms, which have only one defined compartment, have multiple DNA polymerases. Using the appropriate mutants it has been well established that one of these polymerases is the replicative enzyme while the others are involved in gap-tilling or DNA-repair process (Kornberg, 1980). In eukaryotes, DNA polymerases are found in the nuclear, mitochondrial, and in the case of plants, in the chloroplastic fractions. The best studied enzymes are DNA polymerases purified and characterized from animal cells. At this point we feel it appropriate to make a short summary of the properties of animal DNA polymerases as an introduction to the discussion on plant cell DNA polymerases (for reviews on animal DNA polymerases see Weissbach, 1981; Hubscher, 1984; Campbell, 1986; Fry and Loeb, 1986). Animal DNA polymerases Table 1 summarizes the properties of DNA polymerases a, fl, 3' and 8 purified and characterized from animal cells. Much evidence indicates that DNA polymerase a is involved in the replication of nuclear DNA, while DNA polymerase fl is active in DNA repair. DNA polymerase T is the only polymerase found in the
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
82 mitochondria where it replicates the organelle genome. This enzyme is also found in the nuclear fraction but its role in the nucleus is not clear; the proposed role of this enzyme in adenovirus replication, based on the similar effect of inhibitors on purified DNA polymerase 3' and the adenovirus DNA synthesizing activity (van der Vliet and Kwant, 1981) has been contradicted by the recent finding of an adenovirus coded DNA polymerase (Field et al., 1984). These results point to the danger of extrapolating on the nature of specific DNA polymerases based solely on the effect of inhibitor or template specificity studies. It has also been proposed that polymerase 3' is involved in the replication of parvovirus (Kollek and Goulian, 1981). This is a virus with a very small genome and most probably a DNA polymerase from the host cell is used to replicate its single-stranded DNA. The last enzyme found, DNA polymerase 8, has been isolated from calf thymus and reticulocytes (Lee et al., 1981). Polymerase 8 shares many of the properties of DNA polymerase a but it is much more resistant to butylphenyl-dGTP than DNA polymerase a and has a 3'-5' exonuclease associated on the same peptide carrying the polymerase catalytic subunit. The strong association with an exonuclease supports a proofreading activity of this enzyme. A general trend of modem biochemistry is to adopt, for the sake of simplicity, a common nomenclature for enzyme and protein factors from different sources. All kinds of nomenclatures can be found in the literature for DNA polymerases from unicellular algae and higher plants, such as A, B, C. . . . ; I, II, I I I . . . ; 1, 2, 3 etc. With the aim of simplifying the situation, the Greek nomenclature will be used in this review to describe plant DNA polymerases. However, the advance in the knowledge of animal DNA polymerases is considerable when compared with plant enzymes and caution must be taken when extrapolating the properties of animal enzymes a, fl and 7 to the plant DNA polymerases.
DNA polymerases in higher plants It is a difficult and controversial task to classify plant DNA polymerases following the same criteria used with the animal enzymes. The 3 major classes of animal cell DNA polymerases, a, fl and ~,, have proved to be applicable to a wide range of species in the animal kingdom. Nevertheless, DNA polymerases isolated from lower eukaryotes or plants do not fill completely the properties of their animal counterparts.
DNA polymerases from unicellular algae As mentioned in a recent review on the enzymology of nuclear replication in plants (Bryant, t982), a clear distinction can be made between D N A polymerases studied in unicellular algae and
DNA polymerase a In Table 3 we have summarized the properties of some of the high molecular weight DNA polymerases which have been described in plant systems. All these polymerases have a high molecular
lower fungi and those from higher plants. In this review we will not discuss DNA polymerases from lower fungi and we address the reader to an excellent and recent review on this subject (Campbell, 1986). In Table 2 we have summarized the properties of DNA polymerases from 3 unicellular algae, Chlamydomonas (Ross and Harris, 1978a, b), Euglena (McLennan and Keir, 1975a, b) and Chlorella (Aoshima et al., 1982, 1984). All these enzymes seem to have high molecular weight, a characteristic of DNA polymerase a. Nevertheless, as the studies with Chlamydomonas and Euglena are quite old, no specific inhibitors of DNA polymerase were used and it is possible, given the difficulties in separating nuclei and chloroplasts by subcellular fractionation, that one of the two DNA polymerases described in these organisms is the chloroplastic enzyme. Only in the case of Chlorella, aphidicolin, a specific inhibitor of DNA polymerase a from all eukaryotic cells (see below), has been used and shown to inhibit one of the polymerases, the other enzyme characterized in Chlorella seems to correspond to the chloroplast DNA polymerase. The presence of nuclease activity associated with some of the algal DNA polymerases must be taken with caution, since those polymerases have not been purified to apparent homogeneity. Thus, both activities may reside in different proteins which copurify through the different chromatographic steps.
83 TABLE 1 PROPERTIES OF ANIMAL DNA POLYMERASES Properties
a
fl
7
8
Subcellular localisation
Nuclear
Nuclear
Mitochondrial Nuclear
Nuclear
Proposed function
Nuclear DNA replication
DNA repair
Mitochondrial DNA replication
DNA repair
M.W. holoenzyme (kd) M.W. core enzyme (kd)
150-1000 130-170
45 45
> 110
250-290 122
100 0-5 Yes
10 100 No
2 10 Yes
No
Yes
Yes
Yes
Yes
No Yes
No No
No Yes
No No
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes No Yes Yes Yes
No Yes No No No
Yes Yes No No No
Yes
Relative activity Growing cells Quiescent cells Association with primase
Templates Activated DNA Natural RNA templatedeoxy primer DNA template-RNA primer Synthetic R N A templatedeoxy primer Synthetic DNA templatedeoxy primer
Inhibitors NEM ddTTP araCTP Aphidicolin Butyl-dGTP
weight and are strongly inhibited by N-ethylmaleimide (NEM), an SH-reagent which decreases markedly the activity of animal DNA polymerase a. In the case of wheat, spinach, rice and cauliflower, aphidicolin, a specific inhibitor of DNA
Yes No
polymerase a from higher and lower eukaryotes (Spadari et al., 1982), has been shown to inhibit the high molecular weight DNA polymerase. The mechanism of inhibition by aphidicolin seems to be the same as in animal cells: competitive inhibi-
TABLE 2 DNA POLYMERASES FROM UNICELLULAR ALGAE Organism
Polymerase
M.W. (kd)
S
Optima KCI (mM)
Chlamydomonas reinhardii
A
100
5.3
B
200
8-10
Euglena gracilis
A B
190 240
Chlorella ellipsoidea
I II
8.7 10.3 6.1 6.6
50
Inhibition by Mn 2+ (mM)
200
0.2 0.2
25 25
0.2 0.2
60-300 80-200
5 5
Mg 2+ (mM) 2 2
10 20
Aphidicolin
Nuclease
pH
NEM
8.5 7.5
Yes Yes
No No
No Yes
7.3 7.3
Yes Yes
No No
No Yes
8.7 8.0
Yes Yes
Yes No
Use of poly rA-dT
Yes Yes
Sp inacea oleracea (spinach) Oryza sativa (Rice) Brassica oleracea (cauliflower)
7 7 7 9.4
229
6 7
130 ( C n ) 160 180
105
Vinca rosea (Periwinkle) Nicotiana tabacum (tobacco) Triticum (wheat) (a) (b)
6.3
113 100-240
Beta vulgaris (Beet) Pisum sativum (Pea)
Sed. coef. (S)
210-240 110 (B)
Mol. wt. (kd)
Species
50
0 0 0
50 50 50 0
0-25
5
5 10 5-10
6-15 7 5 5
15 15
7.5
8.0 7.5 7.2
7.5 8.3 7.6 7.0
7.5 8.1
Yes
Yes Yes Yes
Yes
Yes Yes
Yes Yes
Yes Yes Yes
Yes
Aphidicolin
NEM
pH
KCI (mM)
MgC12 (mM)
Inhibition
Optima
D N A POLYMERASES a - L I K E F R O M H I G H E R P L A N T S
TABLE 3
No
Yes (only with MnCI 2) Yes (at 30 ° C) No No
No No
U s e polyrA-dT
No No Exo
Exo
Exo
No
Nuclease
F u k a s a w a et al. (1980)
Castroviejo et al. (1979) M i s u m i a n d Weissbach (1982) Amileni et al. (1979)
T y m o n k o a n d D u n h a m (1977) Stevens and Bryant (1978) Chivers and Bryant (1983) G a r d n e r a n d K a d o (1976) Srivastava (1974) Mory et al. (1974) Castroviejo et al. (1979)
Reference
85 i
o
--o--O
4
-o
C~
o
.o
12 DNA polymerose
20 (pg)
Fig. 1. Utilization of poly rC-oligo dO as template by wheat D N A polymerases C I and C u.
tion with dCTP and non-competitive with the other dNTP precursors. This drug has been successfully used to synchronize animal and plant cells in culture (Galli and Sala, 1983). It is interesting to point out that an aphidicolin-inactivating activity has been found in several plant extracts, this activity may interfere with the search for aphidicolin-sensitive DNA polymerases, as well as with the synchronization of plant cells (Sala et al., 1983). Animal DNA polymerase a does not recognize a poly rA-oligo dT template-primer; similar resuits are observed in the case of some of the enzymes presented in Table 3. However, the wheat DNA polymerases B and CII which had been reported inactive with this template (Castroviejo et al., 1979) proved to use poly rA-oligo dT very efficiently when the temperature of the incubation was descreased or a primer-template stabilizing agent was used (Castroviejo et al., 1982). Several parameters must be investigated before concluding that a specific template is not used by a given DNA polymerase. As seen in Fig. 1 the enzymetemplate ratio (in this case wheat DNA polymerase CI) may be a very important factor in the use of a defined template. The subcellular localisation of animal DNA polymerase a has been a controversial subject but it is generally accepted that the bulk of this activity is localized in the nuclear fraction. The same type of studies have not been performed in plants given the difficulty in purifying nuclei with good yields from these organisms. However, using an autoradiographic approach it has been shown that the aphidicolin-sensitive DNA polymerase from rice cells is found exclusively in the nucleus (Sala
et al., 1981). Nuclei purified from soybean cells grown in culture replicates preferentially repeated DNA sequences (Caboche and Lark, 1981). DNA synthesis in soybean nuclei is resistant to dideoxyTTP (ddTTP) which is an inhibitor of DNA polymerase fl and 7 but not of polymerase a. Preliminary results with wheat purified nuclei indicate the presence of a DNA polymerase strongly inhibited by aphidicolin (S. Litvak, unpublished results). It is not clear if this activity corresponds to one or both wheat DNA polymerases sensitive to this drug (enzymes B and CII). The presence of exonuclease activity has been reported in the case of DNA polymerases a-like from periwinkle, wheat and rice. These enzymes have not been purified to apparent homogeneity and the presence of an exonuclease can be ascribed to a copurification event. Aphidicolin has been shown to inhibit a DNA polymerase activity from turnip (Dunham and Bryant, 1986) but the size of the enzyme has not been determined. DNA polymerases from rice and carrot have been submitted to "activity gel" electrophoresis. This technique allows the direct visualization of the catalytic activities of DNA polymerase after SDS-gel electrophoresis, protein renaturation and DNA polymerase assay (Spanos and Hubscher, 1982). Results with plant polymerases indicate the presence of two protein bands with molecular weights of 70 and 110 kd, both carrying DNA polymerase activity (Scovassi et al., 1982).
DNA polymerase fl Much evidence has been accumulated in recent years concerning the existence of an a-type DNA polymerase in plant cells. It is more difficult to make such an assertion in the case of DNA polymerase ft. The 1975 Asilomar meeting on the nomenclature of vertebrate DNA polymerase (Weissbach, 1981) defined DNA polymerase fl as a small molecular weight DNA polymerase confined to the nuclear compartment and highly resistant to NEM. Later it was found that the enzyme was able to use poly rA-dT as templateprimer under certain conditions, was resistant to aphidicolin and inhibited by dideoxyTTP. Low molecular weight DNA polymerases have been found in beet (Tymonko and Dunham, 1977), pea (Stevens et al., 1978; Chivers and Bryant, 1983),
86 tobacco (Srivastaval 1974) and wheat (Castroviejo, unpublished results). A phylogenetic study of DNA polymerase /3 (Chang, 1975) showed the absence of this enzyme in two plants: periwinkle and wheat. The presence of the fl enzyme was followed both by sucrose-gradient centrifugation and the effect of NEM on the low molecular weight peak showing DNA polymerase activity. Results of this author in the case of wheat can be explained by the fact that the low molecular weight DNA polymerase is obtained after an inhibitor is eliminated by phosphocellulose chromatography and only if protease inhibitors are added during the purification procedure (M. Castroviejo, unpublished results). The plant/3-like polymerases have a molecular weight of approximately 50 kd. A 70-kd polymerase with some of the properties of a /3 polymerase has been isolated from cauliflower inflorescences (Fukasawa, 1980b). When considering the size of a DNA polymerase it is important to recall the proteolytic activity present in plant extracts. A catalytically active fragment of 12 kd of spinach DNA polymerase a was obtained when the purification was performed in the absence of protease inhibitors (Misumi and Weissbach, 1982). In the case of wheat, a 50-kd DNA polymerase has been purified to apparent homogeneity. Both the effect of inhibitors and the template specificity were clearly different compared with the two a-like DNA polymerases purified from wheat (Castroviejo et al., 1979, 1982). However, an antibody raised against this tow molecular weight DNA polymerase cross-reacted with DNA polymerase CII (a-type) from wheat (Castroviejo, unpublished results). The effect of NEM shows that the beet and pea small DNA polymerases are only inhibited at concentrations higher than 1 mM and seem to be more resistant than a-like DNA polymerase from the same source. Animal DNA polymerase a is resistant up to 20 mM NEM. Pea /3-like DNA polymerase is strongly bound to chromatin, as is the case of some animal DNA polymerases/3, but no evidence has been obtained that this association has a functional role and it is not explained by an unspecific binding. A chromatin-bound polymerase has also been found in developing soybean (D'Alessandro et al., 1980).
The role of plant /3-like DNA polymerases in DNA repair has not been proved. However, results have been reported that the UV light-induced DNA-repair synthesis in protoplasts of Nieotiana sylvestris is resistant to aphidicolin and thus it is not performed by an a-like DNA polymerase (Sala et al., 1982).
DNA polymerase The discovery of reverse transcriptase, the RNA-dependent DNA polymerase of retrovirus (Temin and Baltimore, 1972), led to the search for a similar enzyme in uninfected cells. A DNA polymerase able to recognize very efficiently a poly rA-oligo dT template was found in human cultured cells (Fridlender et al., 1972). This enzyme, called DNA polymerase 3', was different from the already described animal DNA polymerases a and /3 but it was also different from reverse transcriptase in that it was not able to copy a natural RNA template which is the main property of the retroviral DNA polymerase. DNA polymerase ~, from animal cells is found in the nuclear and mitochondrial fractions. It is the only DNA polymerase involved in the replication of the animal organellar genome (Weissbach, 1981). A similar enzyme of 100 kd was characterized from wheat (Tarrago-Litvak et al., 1975). The wheat enzyme, called DNA polymerase A (Castroviejo et al., 1979), resembles animal DNA polymerase 3' in its exquisite efficiency with poly rA-oligo dT template, as well as by the KC1 stimulation, the strong inhibitory effect of ddTTP and ethidium bromide and the resistance to aphidicolin. Nevertheless, the wheat DNA polymerase ~, is not localized in plant mitochondria and its role has not been elucidated; it is interesting to point out that this enzyme is able to recognize, although to a low extent, a natural RNA template like turnip yellow mosaic virus RNA and avian myeloblastosis virus RNA (Litvak et al., 1984). Wheat DNA polymerase A (3,-like) is also able to initiate DNA synthesis from an RNA primer, a property which differentiates this enzyme from the two a-like polymerases from the same source. Moreover, this enzyme is found associated with a DNA primase activity described in wheat embryos (Graveline et al., 1984). A DNA polymerase able to use poly rA-oligo dT template has been found
87 in healthy turnip cells (Volovitch et al., 1984; Dunham and Bryant, 1986). The spinach chloroplastic DNA polymerase has also been described as a v-like enzyme (see below). A 3-kd protein factor able to inhibit specifically the activity of a DNA polymerase isolated from cauliflower inflorescence, when the template poly rA-oligo dT is used, has been reported (Chou et al., 1981).
Chloroplast DNA polymerase Extranuclear plant cell organelles, chloroplast and mitochondria, contain their own genetic information which is replicated, transcribed and translated inside the organelles. Chloroplast contains a circular DNA of 130-150 kbp (Bohnert et al., 1982). The mechanism of replication or the chloroplastic DNA has not been elucidated but some information, gathered by electron microscopy studies, suggests that the mechanism can be by D-loop displacement, very similar to that described in animal mitochondria; "the rolling circle" mechanism has been also evoked in the replication or chloroplast DNA (Tewari, 1979). Isolated purified chloroplasts are able to synthesize chloroplast DNA in the presence of the appropriate precursors and Mg 2+ (Zimmermann and Weissbach, 1982). Lack of effect of some inhibitors like ddTTP and ethidium bromide on DNA synthesis in whole chloroplasts may be due to the lack of transport of these inhibitors inside the chloroplasts, since the same agents proved to be very efficient when organdie DNA synthesis was followed in chloroplast extracts from maize and liverwort suspension cultures (Zimmermann and Weissbach, 1982; Tanaka et al., 1984). Petunia chloroplast DNA synthesis in organello, however, was shown to be inhibited by ethidium bromide and ddTTP, these results may be explained by the non-integrity of petunia chloroplasts (Overbeeke et al., 1984). Aphidicolin does not inhibit DNA synthesis in whole chloroplasts or organelle extracts in all plant systems studied up to now. Using the technique of molecular hybridization it was shown that the chloroplast genome is somewhat amplified during growth of tobacco cells in the light (Cannon et al., 1985). The first attempt to characterize a chloroplast DNA polymerase was performed a long time ago (Spencer and Whitfeld, 1969). These authors
studied DNA synthesis in purified chloroplasts and described the solubilization of a DNA polymerase able to accept exogenous DNA as template. More recently DNA polymerases from purified chloroplasts of two plants have been isolated and characterized. The enzyme from spinach organelles (Sala et al., 1980) has been classified as a v-like DNA polymerase given its high efficiency with poly rA-oligo dT as template, while activated DNA, a native DNA which is gapped with very low amounts of pancreatic DNAase I, is almost not recognized by this enzyme. The enzyme has a relative M.W. of 105 kd and its activity is dependent on the presence of KC1 and Mn 2+, it is inhibited by NEM and resistant to aphidicolin. Surprisingly, the DNA polymerase from pea chloroplasts (McKown and Tewari, 1984) does not recognize a poly rA-dT template but it is inhibited, as the spinach polymerase, by NEM, ethidium bromide and is resistant to aphidicolin. The pea polymerase has been purified to apparent homogeneity; it is a monomeric protein of about 87 kd. No nuclease activity was detected in the purified pea DNA polymerase. It is not clear if the difference in template recognition, observed in the two chloroplast DNA polymerases mentioned above, is due to intrinsic enzymatic differences, to the higher degree of purification of the pea polymerase or to a partial proteolysis of the latter enzyme which would explain the difference of about 20 kd in the molecular weight of both enzymes.
Mitochondrial DNA polymerase The mitochondrial (mt) genomes of animal and plants differ in many respects, although the physiological function of mitochondria from all sources seems to be the same. The differences include size, structure and complexity, gene structure and codon usage (Pring and Lonsdale, 1985; Bendich, 1985). A striking finding that exemplifies the complexity of the plant mt genome is the size range that can vary from 218 kbp in turnip to about 2500 in watermelon. Surprisingly the green alga Chlamydomonas has a mitochondrial genome of about 16 kbp. The main feature of higher plant mt is the presence of a master, circular chromosome and subgenomic circular molecules generated by recombination between directly repeated elements
88 of the genome (Mulligan and Walbot, 1986). The recent interest in the study of plant mt genome organization can be explained by the observation, 10 years ago, that alterations in the pattern of restriction fragments of mt DNA, but not chloroplast DNA, correlated with cytoplasmically inherited male sterility. It is interesting to point out in this respect the presence of plasmid-like DNAs in the mitochondria of some maize cytoplasmic male sterile strains. The genomic structure of these plasmid-like DNAs differs from that of the mt DNA since they are linear and have a covalently bound protein at their 5' end; thus, they resemble the structure of some viral genomes such as q~29 and adenovirus (Laughan and Gabay-Laughan, 1983). As in the case of chloroplasts, isolated plant mitochondria are able to synthesize mt DNA (Ricard et al., 1983; Bedinger and Walbot, 1986). The mitochondrial genome is extensively labelled although no direct evidence of DNA initiation is presented. DNA synthesis in wheat mitochondria is strongly inhibited by NEM, ethidium bromide and ddTTP and it is resistant to aphidicolin. ATP stimulates the incorporation of dNTP precursors into mt DNA in wheat organelles, while it inhibits DNA synthesis in maize mitochondria. Nitrofurantoin, a potent inhibitor of bacterial growth, has proved to decrease strongly DNA synthesis in mitochondria from 48 h etiolated Vigna sinensis seedlings (Palit et al., 1980). A mt lysate has been obtained from wheat mitochondria and the DNA synthesizing properties of the system have been described (Ricard et al., 1983). DNA synthesis involves the action of a DNA primase activity confined to the mt compartment which synthesizes the RNA primers used by the mt DNA polymerase to initiate organelle DNA synthesis (M. Echeverria, unpublished results). A DNA polymerase with an apparent native molecular weight of about 180 kd has been solubilized, purified and characterized from wheat mitochondria (Castroviejo et al., 1979; Christophe et al., 1981). The enzyme is clearly distinct from the other DNA polymerases described in wheat and also differs from the animal mt DNA polymerase in the inability to use, with manganese or magnesium ions, a poly rA-oligo dT template, which is the preferred template of the animal
enzyme. Thus, the plant mitochondrial DNA polymerase cannot be classified as a T-like polymerase. A similar enzyme has been found in the mitochondria of rice cells (F. Sala, unpublished results). However, the strong effect of inhibitors like ddTTP, ethidium bromide and NEM, as well as the resistance to aphidicolin, seem to be similar in mt DNA polymerases from all eukaryotic cells studied up to now. A preliminary report on the mt DNA polymerase from cauliflower inflorescence has also been published (Fukasawa and Chou, 1980a).
Plant viral DNA polymerases The caulimoviruses are the only group of plant viruses known to contain double stranded DNA. The type member of the group, cauliflower mosaic virus (CaMV), is by far the most studied (Hohn et al., 1982). In parallel with the studies aimed at using this virus as an eukaryote vector in plant genetic engineering, several groups became involved in the study of the expression of the CaMV genome. One of the biggest surprises of these last years in the domain of plant molecular biology was the discovery that CaMV, although a DNA virus, uses RNA as a replicative intermediate and employs a reverse transcriptase for genome replication (Pfeiffer and Hohn, 1983; Hull, 1984; Hohn et al., 1985). This situation, the involvement of a reverse transcriptase, is similar to that of the replication of hepatitis B virus which has also a DNA genome and of the retroviruses which have an RNA genome. It is generally accepted that the DNA polymerase which replicates CaMV is, at least in part, coded by gene V product (ORF V) of the viral genome. The determination of the whole sequence of CaMV DNA allowed the finding that significant homologies exist between the gene V product and reverse transcriptase from hepatitis B virus and retroviruses (Volovitch et al., 1984; Toh et al., 1984). Based also on the sequence of CamV DNA it has been proposed that a methioninespecific tRNA may be involved in the priming of the reverse transcriptase reaction, in a similar way to the mechanism of retroviruses replication. The enzymological characterization of the DNA polymerase involved in CamMV DNA replication has been hampered by the low level and instability of the enzyme, as well as by the background
89 activities of the host D N A polymerases. A polymerase activity, very active with poly rA and poly rC as templates, was observed in turnip infected leaves. The activity could be chromatographically separated from D N A polymerase 3' (Volovitch et al., 1984). Other authors have been unable to detect differences at the level of D N A polymerases in healthy and CaMV infected turnip (Dunham and Bryant, 1986). The presence of a reverse transcriptase activity has been observed in viral replication complexes obtained by hypotonic shocks of viral infected organelles. CaMV D N A synthesis is insensitive to aphidicolin but partially inhibited by ddTTP (Pfeiffer et al., 1984). The same authors used the "activity gel" technique to characterize the CaMV D N A polymerase after polyacrylarnide gel electrophoresis in the presence of sodium dodecyl sulphate and protein renaturation (Spanos and Hubscher, 1982). The enzyme involved in viral replication coincided with a band of 75 kd, while an aphidicolin-sensitive D N A polymerase, present in healthy and infected leaves, was shown at the level of a l l 0 - k d peptide. A low but significant D N A polymerase activity has been detected within the CaMV particle; the molecular weight of this polymerase determined by activity gel is 76 kd (Menissier et al., 1984). The D N A polymerase activity associated to CaMV-replicating complexes was specifically inhibited by immune serum raised against a synthetic peptide corresponding to a portion of the viral gene V (ORF V) protein product (Laquel et al., 1986). The gene coding for ORF V has been cloned and expressed in the yeast Saccharomyces cerevisiae. Yeast expressing this gene accumulated significant levels of reverse transcriptase activity (Takatsuji et al., 1986). These and the previous results suggest strongly that CaMV code for a DNA polymerase different from the host DNA polymerases and able to act as a reverse transcriptase. Information on the replication of other plant D N A viruses is very scarce. Some results concerning the geminivirus have been presented (Coutts and Buck, 1985). The geminiviruses are a group of plant viruses whose members are characterized by the possession of twin isometric particles and genomes of single-stranded D N A circles between 2.5 and 2.7 kb. Nuclei and nuclear extracts from tomato golden mosaic virus (TGMV) infected
tobacco leaves were used for in vitro DNA synthesis. The synthesis of virus-specific D N A was resistant to aphidicolin, sensitive to N E M and ddTTP and stimulated by KC1 and ATP. T G M V is a two-component geminivirus (A and B). No component alone can be replicated when inoculated on tobacco leaves. In a recent study (Rogers et al., 1986) multimers of the A or the B component of T G M V were cloned separately in the T-arm of a disarmed Ti plasmid. Infection and regeneration of Petunia leaf discs led to no symptoms. When the plants containing the A or B components were crossed, however, plants showing T G M V infection symptoms were recovered with the expected frequency. Interestingly, plants with the A component alone (but not the B component) showed, by Southern blotting, replication of single- and double-stranded viral DNA. These results suggest strongly that viral DNA polymerase functions (the enzyme itself or a factor needed for a host polymerase) were encoded by the A component.
Acknowledgments Work performed in the authors laboratory was supported by C.N.R.S., the University of Bordeaux II and N.A.T,O. The authors are grateful to many colleagues for providing reprints and preprints of research articles and to Dr. L. Tarrago-Litvak for helpful comments on the manuscript.
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90 D. Boulter (Eds.), Encyclopedia of Plant Physiology, Vol. 14B, Springer, Heidelberg, pp. 475-530, Bryant, J.A. (1982) DNA replication and the cell cycle, in: B. Partnier and D. Boulter (Eds.), The Encyclopedia of Plant Physiology, 14B, Springer, Heidelberg, pp. 75-110. Caboche, M., and K.G. Lark (1981) Preferential replication of repeated DNA sequences in nuclei isolated from soybean cells grown in culture, Proc. Natl. Acad. Sci. (U.S.A.), 78, 1731-1735. Campbell, J.L. (1986) Eukaryotic DNA replication, Annu. Rev. Biochem., 55, 733-771. Cannon, G., S. Heinhorst, J. Siedlecki and A. Weissbach (1985) Chloroplast DNA synthesis in light and dark grown cultured Nicotiana tabacurn cells as determined by molecular hybridization, Plant Cell Rep., 4, 41-45. Castroviejo, M., L. Tarrago-Litvak and S. Litvak (1975) Partial purification and characterization of two cytoplasmic DNA polymerases from ungerminated wheat, Nucleic Acids Res., 2, 2077-2090. Castroviejo, M., D. Tharaud, L. Tarrago-Litvak and S. Litvak (1979) Multiple DNA polymerases from quiescent wheat embryos, Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria, Biochem., J., 181, 183-191. Castroviejo, M., P.V. Graves, D. Tharaud, E. Hevia-Campos and S. Litvak (1982) Etnidium bromide stimulation of DNA polymerase activity by stabilization of the primertemplate duplex, Biochimie, 64, 195-202. Chang, L.M.S. (1976) Phylogeny of polymerase fl, Science, 191, 1183-1185. Cnivers, H.J., and J.A. Bryant (1983) Molecular weights of the DNA polymerases in a nigher plant, Pisum sativum, Biochem. Biophys. Res. Commun., 110, 632-639. Chou, M.-Y., H. Matsumoto, H. Fukata and H. Fukasawa (1981) Template-specificity inhibitor for DNA polymerase isolated from cauliflower inflorescence, Biocnim. Biophys. Acta, 652, 48-54. Christophe, L., L. Tarrago-Litvak, M. Castroviejo and S. Litvak (1981) Mitochondrial DNA polymerase from wheat embryos, Plant Sci. Lett., 21, 181-192. Coutts, R.H.A., and K.W. Buck (1985) DNA and RNA polymerase activities of nuclei and hypotonic extracts of nuclei isolated from TGMV infected tobacco leaves, Nucleic Acids Res., 13, 7881-7897. D'Alesandro, M.M., R.H. Jaskot and V.L. Dunham (1980) Soluble and chromatin-bound DNA polymerases in the developing soybean, Biochem. Biophys. Res. Commun., 94, 233-239. Dunham, V.L., and J.A. Bryant (1986) DNA polymerase activities in healthy and CaMV-infected turnip (Brassica rapa) plants, Ann. Botany, 57, 81-86. Field, J., R.M. Gronostajski and J. Hurwitz (1984) Properties of the adenovirus DNA polymerase, J. Biol. Chem. 259, 9487-9495. Fridlender, B., M. Fry, A. Bolden and A. Weissbach (1972) A new synthetic RNA-dependent DNA polymerase from human tissue culture cells, Proc. Natl. Acad. Sci. (U.S.A.), 69, 452-455.
Fry, M., and L.A. Loeb (1986) Animal Cell DNA Polymerases, CRC Press. Fukasawa, H., and M.-Y. Chou (1980a) Mitochondrial DNA polymerase from cauliflower inflorescence, Jpn. J. Genet., 55,441-445. Fukasawa, H., M. Yamaguchi, M.-Y. Chou, H. Matsumoto and A. Matsukage (1980b) Characterization of two DNA polymerases from cauliflower inflorescence, J. Biochem., 87, 1167-1175. Galli, M.G., and F. Sala (1983) Apnidicolin as synchronizing agent in root tip meristems of Haplopappus gracilis, Plant Cell Rep., 2, 156-159. Gardner, J.M., and C.I. Kado (1976) High molecular weight DNA polymerase from crown gall tumor cells of periwinkle (Vinca rosea), Biochemistry, 15, 688-697. Graveline, J., S. Tarrago-Litvak, M. Castroviejo and S. Litvak (1984) DNA primase activity from wheat embryos, Plant Mol. Biol., 3, 207-215. Hohn, T., K. Richards and G. Lebeurier (1982) Cauliflower mosaic virus on its way to becoming a useful plant vector. Curr. Top. Microbiol. Immunol., 96, 193-236. Hohn, T., B. Hohn and P. Pfeiffer (1985) Reverse transcription in CaMV, Trends Biochem. Sci., 10, 205-209. Hubscher, U. (1984) DNA polymerase holoenzymes, Trends Biochem. Sci., 9, 390-393. Hull, R. (1984) A model for the expression of CaMV nucleic acid, Plant Mol. Biol., 3, 121-125. Kollek, R., and M. Goulian (1981) Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma, Proc. Natl. Acad. Sci. (U.S.A.), 78, 6206-6210. Kornberg, A. (1980, suppl. 1982) DNA Replication, Freeman, San Francisco. Laquel, P., V. Ziegler and L. Hirth (1986) The 80K polypeptide. associated with the replication complexes of CaMV is recognized by antibodies to gene V translation product, J. Gen. Virol., in press. Laughnan, J.R., and S. Gabay-Laughnan (1983) Cytoplasmic male sterility in maize, Annu. Rev. Genet., 17, 27-48. Lee, M., C. Tan, K. Downey and A. So (1981) Structural and functional properties of calf thymus DNA polymerase 8, Progress in Nucleic Acids Res. Mol. Biol., 26, 83-96. Litvak, S., J. Graveline, L. Zourgui, P. Carvallo, A. Solari, H. Aoyama, M. Castroviejo and L. Tarrago-Litvak (1984) Studies on the initiation of DNA synthesis in plant and animal cells, in: Proteins Involved in DNA Replication, Adv. Exptl. Med. Biol., Vol. 179, Plenum, New York, pp. 249-262. McKown, R.L., and K.K. Tewari (1984) Purification and properties of pea chloroplast DNA polymerase, Proc. Natl. Acad. Sci. (U.S.A.), 81, 2354-2358. McLennan, A.G., and H.M. Keir (1975a) DNA polymerases of Euglena gracilis, Purification and properties of two distinct DNA polymerases of high molecular weight, Biochem., J., 151,227-238. McLennan, A.G., and H.M. Keir (1975b) DNA polymerases of Euglena gracilis, Primer-template utilization and enzyme activities associated with the two DNA polymerases of high molecular weight, Biochem. J., 151,239-247.
91 Menissier, J., P. Laquel, G. Lebeurier and L. Hirth (1984) A DNA polymerase activity is associated with CaMV, Nucleic Acids Res., 12, 8769-8778. Misumi, M., and A. Weissbach (1982) The isolation and characterization of DNA polymerase a from spinach, J. Biol. Chem. 257, 2323-2329. Mory, Y.Y., D. Chen and S. Sarid (1974) DNA polymerase from wheat embryos, Plant Physiol., 53, 377-381. Mulligan, R.M., and V. Walbot (1986) Gene expression and recombination in plant mt genomes, Trends Genet., 2, 263-266. Overbeeke, N., J.H. de Waard and A.J. Kool (1984) Characterization of in vitro DNA synthesis in an isolated chloroplast system of Petunia hybrida, in: Proteins involved in DNA replication, Adv. Exptl. Med. Biol., Vol. 179, Plenum, New York, pp. 107-112. Palit, S., B.B. Goswami and D.K. Dube (1980) The effect of nitrofurantoin on DNA synthesis in plant mitochondria, Biochem., J., 186, 325-329. Pfeiffer, P., and T. Hohn (1983) Involvement of reverse transcription in the replication of CaMV: A detailed model and test of some aspects, Cell, 33, 781-789. Pfeiffer, P., P. Laquel and T. Hohn (1984) CaMV replication complexes: characterization of the associated enzymes and of the polarity of the DNA synthesized in vitro, Plant Mol. Biol., 3, 261-270. Pring, D.R., and D.M. Lonsdale (1985) Molecular biology of higher plant mt DNA, Int. Rev. Cytol., 97, 1-45. Ricard, B., M. Echeverria, L. Christophe and S. Litvak (1983) DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos, Plant Mol. Biol., 2, 167-175. Rogers, S.G., D.M. Bisaro, R.B. Horsch, R.T. Fraley, N.L. Hoffmann, L. Brand, J.S. Elmer and A.M. Lloyd (1986) TGMV A component DNA replicates autonomously in transgenic plants, Cell, 45, 593-600. Ross, C.A., and W.J. Harris (1978a) DNA polymerase from Chlamydomonas reinhardii, Purification and properties, Biochem. J., 171,231-240. Ross, C.A., and W.J. Harris (1978b) DNA polymerase from Chlamydomonas reinhardii, Further characterization, action of inhibitors and associated nuclease activities, Biochem. J., 171,241-249. Sala, F., A.R. Amileni and S. Spadari (1980) A 33-like DNA polymerase in spinach chloroplasts, Eur. J. Biochem., 112, 211-217. Sala, F., M.GI Galli, M. Levi, D. Burroni, B. Parisi, G. Pedrali-Noy and S. Spadari (1981) Functional roles of the plant a-like and 33-like DNA polymerases, FEBS Lett., 124, 112-118. Sala, F., E. Magnien, M.G. Galli, X. Dalschaert, G. Pedrali-Noy and S. Spadari (1982) DNA repair synthesis in plant protoplasts is aphidicolin-resistant, FEBS Lett., 138, 213-217. Sala, F., C. Sala, M.G. Galli, E. Nielsen, G. Pedrali-Noy and S. Spadari (1983) Inactivation of aphidicolin by plant cells, Plant Cell Rep., 2, 265-268. Scovasi, A.I., S. Torsello, P. Plevani, G.F. Badaracco and U. Bertazonni (1982) Active polypeptide fragments common
to prokaryotic, eukaryotic and mitochondrial DNA polymerases, EMBO J., 1, 1161-1165. Spadari, S., F. Sala and G. Pedrali-Noy (1982) Aphidicolin: a specific inhibitor of nuclear DNA replication in eukaryotes, Trends Biochem. Sci., 7, 29-32. Spanos, A., and U. Hubscher (1982) Recovery of functional proteins in sodium dodecyl sulfate gels, Methods in Enzymology, Vol. 91, Academic Press, New York, pp. 263-277. Spencer, D., and P.R. Whitfeld (1969) The characteristics of spinach chloroplast DNA polymerase, Arch. Biochem. Biophys., 132, 477-488. Srivastava, B.I.S. (1974) A 7S DNA polymerase in the cytoplasmic fraction from higher plants, Life Sci., 14, 1947-1954. Stevens, C., and J.A. Bryant (1978) Partial purification and characterization of the soluble DNA polymerase (polymerase a) from seedlings of Pisum sativum L., Planta, 138, 127-132. Stevens, C., J.A. Bryant and P.C. Wyvill (1978) Chromatinbound DNA polymerase from higher plants, Planta, 143, 113-120. Takatsuji, H., H. Hirochika, T. Fukishi and J.-E. Ikeda (1986) Expression of CaMV virus reverse transcriptase in yeast, Nature (London), 319, 240-243. Tanaka, A., Y. Yamano, H. Fukuzawa, K. Ohyama and T. Komano (1984) In vitro DNA synthesis by chloroplasts isolated from Marchantia polymorpha L cell suspension culture, Agr. Biol. Chem., 48, 1239-1244. Tarrago-Litvak, L., M. Castroviejo and S. Litvak (1975) Studies on a DNA polymerase 33-like enzyme from wheat embryos, FEBS Lett., 59, 125-130. Temin, H.M., and D. Baltimore (1972) RNA-directed DNA synthesis and RNA tumor virus, Adv. Virus Res., 17, 129-186. Tewari, K.K. (1979) Chloroplast DNA: Structure, transcription and replication, in: T.C. Hall and J.W. Davies (Eds.), Nucleic Acids in Plants, Vol. 1, CRC Press, pp. 41-110. Toh, H., H. Hayashida and T. Miyata (1983) Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and CamV, Nature (London), 305, 827-829. Tymonko, J.M., and V.L. Dunham (1977) Evidence for DNA polymerase a and fl activity in sugar beet, Physiol. Plant., 40, 27-30. van der Vliet, P.C., and M.M. Kwant (1981) Role of DNA polymerase y in adenovirus DNA replication, Mechanism of inhibition by ddNTP, Biochemistry, 20, 2628-2632. Volovitch, M., N. Modjtahedi, P. Yot and G. Brun (1984) RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves, EMBO J., 3, 309-314. Weissbach, A. (1981) Cellular and viral induced DNA polymerases, The Enzymes, Vol. 14, Academic Press, New York, pp. 67-86. Zimmermann, W., and A. Weissbach (1982) DNA synthesis in isolated chloroplasts and chloroplast extracts of maize, Biochemistry, 21, 3334-3343.
81
MTR04379
D N A polymerases from plant cells * Simon Litvak and Michel Castroviejo Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1, rue Camille Saint Sa~ns, 33077 Bordeaux Cedex (France)
(Received 5 January 1987) (Accepted 11 March 1987)
Keywords: DNA polymerases; Plant cells; DNA primase; Topoisomerase; Helicase; Ribonuclease; Ligase; DNA viruses.
The replication and repair of DNA involve the concerted activity of several protein factors and enzymes. These include DNA polymerases, proteins which are associated to the DNA polymerases, DNA primase, topoisomerase, helicase, DNA-binding proteins, ribonuclease and ligase (for a review see Kornberg, 1980). By far the most studied of these proteins are DNA polymerases which are found in all the cellular compartments where DNA must be duplicated. DNA polymerases catalyse the incorporation of deoxyribonucleoside monophosphates (dNMP) in a 5'-3' direction using the parental strands as templates. These enzymes use deoxynucleoside 5'-triphosphates as substrate; pyrophosphate is eliminated in the reaction and a phosphodiester bond is formed between the remaining phosphate of the dNMP and the 3'-OH group of the previously incorporated precursor. The breakage of the alpha-beta phosphate linkage of the deoxyribonucleoside triphosphate provides the energy for the reaction. The sequence of the daughter strands is determined by the insertion of the complementary base following the classical A-T, G - C rule. No DNA polymerase is able to initiate DNA synthesis on a single-stranded DNA in the ab-
* This article is dedicated to Professor L.F. Leloir (Buenos Aires, Argentina) on the occasion of his 80th birthday. Correspondence: Dr. S. Litvak, Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1, rue Camille Saint SaSns, 33077 Bordeaux Cedex (France).
sence of a primer which provides a free 3'-OH able to form the phosphodiester bond with the oncoming precursor nucleoside. RNA primers for DNA initiation are synthesized in the lagging strand of the replication fork by DNA primase (Kornberg, 1980). Prokaryotic organisms, which have only one defined compartment, have multiple DNA polymerases. Using the appropriate mutants it has been well established that one of these polymerases is the replicative enzyme while the others are involved in gap-tilling or DNA-repair process (Kornberg, 1980). In eukaryotes, DNA polymerases are found in the nuclear, mitochondrial, and in the case of plants, in the chloroplastic fractions. The best studied enzymes are DNA polymerases purified and characterized from animal cells. At this point we feel it appropriate to make a short summary of the properties of animal DNA polymerases as an introduction to the discussion on plant cell DNA polymerases (for reviews on animal DNA polymerases see Weissbach, 1981; Hubscher, 1984; Campbell, 1986; Fry and Loeb, 1986). Animal DNA polymerases Table 1 summarizes the properties of DNA polymerases a, fl, 3' and 8 purified and characterized from animal cells. Much evidence indicates that DNA polymerase a is involved in the replication of nuclear DNA, while DNA polymerase fl is active in DNA repair. DNA polymerase T is the only polymerase found in the
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
82 mitochondria where it replicates the organelle genome. This enzyme is also found in the nuclear fraction but its role in the nucleus is not clear; the proposed role of this enzyme in adenovirus replication, based on the similar effect of inhibitors on purified DNA polymerase 3' and the adenovirus DNA synthesizing activity (van der Vliet and Kwant, 1981) has been contradicted by the recent finding of an adenovirus coded DNA polymerase (Field et al., 1984). These results point to the danger of extrapolating on the nature of specific DNA polymerases based solely on the effect of inhibitor or template specificity studies. It has also been proposed that polymerase 3' is involved in the replication of parvovirus (Kollek and Goulian, 1981). This is a virus with a very small genome and most probably a DNA polymerase from the host cell is used to replicate its single-stranded DNA. The last enzyme found, DNA polymerase 8, has been isolated from calf thymus and reticulocytes (Lee et al., 1981). Polymerase 8 shares many of the properties of DNA polymerase a but it is much more resistant to butylphenyl-dGTP than DNA polymerase a and has a 3'-5' exonuclease associated on the same peptide carrying the polymerase catalytic subunit. The strong association with an exonuclease supports a proofreading activity of this enzyme. A general trend of modem biochemistry is to adopt, for the sake of simplicity, a common nomenclature for enzyme and protein factors from different sources. All kinds of nomenclatures can be found in the literature for DNA polymerases from unicellular algae and higher plants, such as A, B, C. . . . ; I, II, I I I . . . ; 1, 2, 3 etc. With the aim of simplifying the situation, the Greek nomenclature will be used in this review to describe plant DNA polymerases. However, the advance in the knowledge of animal DNA polymerases is considerable when compared with plant enzymes and caution must be taken when extrapolating the properties of animal enzymes a, fl and 7 to the plant DNA polymerases.
DNA polymerases in higher plants It is a difficult and controversial task to classify plant DNA polymerases following the same criteria used with the animal enzymes. The 3 major classes of animal cell DNA polymerases, a, fl and ~,, have proved to be applicable to a wide range of species in the animal kingdom. Nevertheless, DNA polymerases isolated from lower eukaryotes or plants do not fill completely the properties of their animal counterparts.
DNA polymerases from unicellular algae As mentioned in a recent review on the enzymology of nuclear replication in plants (Bryant, t982), a clear distinction can be made between D N A polymerases studied in unicellular algae and
DNA polymerase a In Table 3 we have summarized the properties of some of the high molecular weight DNA polymerases which have been described in plant systems. All these polymerases have a high molecular
lower fungi and those from higher plants. In this review we will not discuss DNA polymerases from lower fungi and we address the reader to an excellent and recent review on this subject (Campbell, 1986). In Table 2 we have summarized the properties of DNA polymerases from 3 unicellular algae, Chlamydomonas (Ross and Harris, 1978a, b), Euglena (McLennan and Keir, 1975a, b) and Chlorella (Aoshima et al., 1982, 1984). All these enzymes seem to have high molecular weight, a characteristic of DNA polymerase a. Nevertheless, as the studies with Chlamydomonas and Euglena are quite old, no specific inhibitors of DNA polymerase were used and it is possible, given the difficulties in separating nuclei and chloroplasts by subcellular fractionation, that one of the two DNA polymerases described in these organisms is the chloroplastic enzyme. Only in the case of Chlorella, aphidicolin, a specific inhibitor of DNA polymerase a from all eukaryotic cells (see below), has been used and shown to inhibit one of the polymerases, the other enzyme characterized in Chlorella seems to correspond to the chloroplast DNA polymerase. The presence of nuclease activity associated with some of the algal DNA polymerases must be taken with caution, since those polymerases have not been purified to apparent homogeneity. Thus, both activities may reside in different proteins which copurify through the different chromatographic steps.
83 TABLE 1 PROPERTIES OF ANIMAL DNA POLYMERASES Properties
a
fl
7
8
Subcellular localisation
Nuclear
Nuclear
Mitochondrial Nuclear
Nuclear
Proposed function
Nuclear DNA replication
DNA repair
Mitochondrial DNA replication
DNA repair
M.W. holoenzyme (kd) M.W. core enzyme (kd)
150-1000 130-170
45 45
> 110
250-290 122
100 0-5 Yes
10 100 No
2 10 Yes
No
Yes
Yes
Yes
Yes
No Yes
No No
No Yes
No No
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes No Yes Yes Yes
No Yes No No No
Yes Yes No No No
Yes
Relative activity Growing cells Quiescent cells Association with primase
Templates Activated DNA Natural RNA templatedeoxy primer DNA template-RNA primer Synthetic R N A templatedeoxy primer Synthetic DNA templatedeoxy primer
Inhibitors NEM ddTTP araCTP Aphidicolin Butyl-dGTP
weight and are strongly inhibited by N-ethylmaleimide (NEM), an SH-reagent which decreases markedly the activity of animal DNA polymerase a. In the case of wheat, spinach, rice and cauliflower, aphidicolin, a specific inhibitor of DNA
Yes No
polymerase a from higher and lower eukaryotes (Spadari et al., 1982), has been shown to inhibit the high molecular weight DNA polymerase. The mechanism of inhibition by aphidicolin seems to be the same as in animal cells: competitive inhibi-
TABLE 2 DNA POLYMERASES FROM UNICELLULAR ALGAE Organism
Polymerase
M.W. (kd)
S
Optima KCI (mM)
Chlamydomonas reinhardii
A
100
5.3
B
200
8-10
Euglena gracilis
A B
190 240
Chlorella ellipsoidea
I II
8.7 10.3 6.1 6.6
50
Inhibition by Mn 2+ (mM)
200
0.2 0.2
25 25
0.2 0.2
60-300 80-200
5 5
Mg 2+ (mM) 2 2
10 20
Aphidicolin
Nuclease
pH
NEM
8.5 7.5
Yes Yes
No No
No Yes
7.3 7.3
Yes Yes
No No
No Yes
8.7 8.0
Yes Yes
Yes No
Use of poly rA-dT
Yes Yes
Sp inacea oleracea (spinach) Oryza sativa (Rice) Brassica oleracea (cauliflower)
7 7 7 9.4
229
6 7
130 ( C n ) 160 180
105
Vinca rosea (Periwinkle) Nicotiana tabacum (tobacco) Triticum (wheat) (a) (b)
6.3
113 100-240
Beta vulgaris (Beet) Pisum sativum (Pea)
Sed. coef. (S)
210-240 110 (B)
Mol. wt. (kd)
Species
50
0 0 0
50 50 50 0
0-25
5
5 10 5-10
6-15 7 5 5
15 15
7.5
8.0 7.5 7.2
7.5 8.3 7.6 7.0
7.5 8.1
Yes
Yes Yes Yes
Yes
Yes Yes
Yes Yes
Yes Yes Yes
Yes
Aphidicolin
NEM
pH
KCI (mM)
MgC12 (mM)
Inhibition
Optima
D N A POLYMERASES a - L I K E F R O M H I G H E R P L A N T S
TABLE 3
No
Yes (only with MnCI 2) Yes (at 30 ° C) No No
No No
U s e polyrA-dT
No No Exo
Exo
Exo
No
Nuclease
F u k a s a w a et al. (1980)
Castroviejo et al. (1979) M i s u m i a n d Weissbach (1982) Amileni et al. (1979)
T y m o n k o a n d D u n h a m (1977) Stevens and Bryant (1978) Chivers and Bryant (1983) G a r d n e r a n d K a d o (1976) Srivastava (1974) Mory et al. (1974) Castroviejo et al. (1979)
Reference
85 i
o
--o--O
4
-o
C~
o
.o
12 DNA polymerose
20 (pg)
Fig. 1. Utilization of poly rC-oligo dO as template by wheat D N A polymerases C I and C u.
tion with dCTP and non-competitive with the other dNTP precursors. This drug has been successfully used to synchronize animal and plant cells in culture (Galli and Sala, 1983). It is interesting to point out that an aphidicolin-inactivating activity has been found in several plant extracts, this activity may interfere with the search for aphidicolin-sensitive DNA polymerases, as well as with the synchronization of plant cells (Sala et al., 1983). Animal DNA polymerase a does not recognize a poly rA-oligo dT template-primer; similar resuits are observed in the case of some of the enzymes presented in Table 3. However, the wheat DNA polymerases B and CII which had been reported inactive with this template (Castroviejo et al., 1979) proved to use poly rA-oligo dT very efficiently when the temperature of the incubation was descreased or a primer-template stabilizing agent was used (Castroviejo et al., 1982). Several parameters must be investigated before concluding that a specific template is not used by a given DNA polymerase. As seen in Fig. 1 the enzymetemplate ratio (in this case wheat DNA polymerase CI) may be a very important factor in the use of a defined template. The subcellular localisation of animal DNA polymerase a has been a controversial subject but it is generally accepted that the bulk of this activity is localized in the nuclear fraction. The same type of studies have not been performed in plants given the difficulty in purifying nuclei with good yields from these organisms. However, using an autoradiographic approach it has been shown that the aphidicolin-sensitive DNA polymerase from rice cells is found exclusively in the nucleus (Sala
et al., 1981). Nuclei purified from soybean cells grown in culture replicates preferentially repeated DNA sequences (Caboche and Lark, 1981). DNA synthesis in soybean nuclei is resistant to dideoxyTTP (ddTTP) which is an inhibitor of DNA polymerase fl and 7 but not of polymerase a. Preliminary results with wheat purified nuclei indicate the presence of a DNA polymerase strongly inhibited by aphidicolin (S. Litvak, unpublished results). It is not clear if this activity corresponds to one or both wheat DNA polymerases sensitive to this drug (enzymes B and CII). The presence of exonuclease activity has been reported in the case of DNA polymerases a-like from periwinkle, wheat and rice. These enzymes have not been purified to apparent homogeneity and the presence of an exonuclease can be ascribed to a copurification event. Aphidicolin has been shown to inhibit a DNA polymerase activity from turnip (Dunham and Bryant, 1986) but the size of the enzyme has not been determined. DNA polymerases from rice and carrot have been submitted to "activity gel" electrophoresis. This technique allows the direct visualization of the catalytic activities of DNA polymerase after SDS-gel electrophoresis, protein renaturation and DNA polymerase assay (Spanos and Hubscher, 1982). Results with plant polymerases indicate the presence of two protein bands with molecular weights of 70 and 110 kd, both carrying DNA polymerase activity (Scovassi et al., 1982).
DNA polymerase fl Much evidence has been accumulated in recent years concerning the existence of an a-type DNA polymerase in plant cells. It is more difficult to make such an assertion in the case of DNA polymerase ft. The 1975 Asilomar meeting on the nomenclature of vertebrate DNA polymerase (Weissbach, 1981) defined DNA polymerase fl as a small molecular weight DNA polymerase confined to the nuclear compartment and highly resistant to NEM. Later it was found that the enzyme was able to use poly rA-dT as templateprimer under certain conditions, was resistant to aphidicolin and inhibited by dideoxyTTP. Low molecular weight DNA polymerases have been found in beet (Tymonko and Dunham, 1977), pea (Stevens et al., 1978; Chivers and Bryant, 1983),
86 tobacco (Srivastaval 1974) and wheat (Castroviejo, unpublished results). A phylogenetic study of DNA polymerase /3 (Chang, 1975) showed the absence of this enzyme in two plants: periwinkle and wheat. The presence of the fl enzyme was followed both by sucrose-gradient centrifugation and the effect of NEM on the low molecular weight peak showing DNA polymerase activity. Results of this author in the case of wheat can be explained by the fact that the low molecular weight DNA polymerase is obtained after an inhibitor is eliminated by phosphocellulose chromatography and only if protease inhibitors are added during the purification procedure (M. Castroviejo, unpublished results). The plant/3-like polymerases have a molecular weight of approximately 50 kd. A 70-kd polymerase with some of the properties of a /3 polymerase has been isolated from cauliflower inflorescences (Fukasawa, 1980b). When considering the size of a DNA polymerase it is important to recall the proteolytic activity present in plant extracts. A catalytically active fragment of 12 kd of spinach DNA polymerase a was obtained when the purification was performed in the absence of protease inhibitors (Misumi and Weissbach, 1982). In the case of wheat, a 50-kd DNA polymerase has been purified to apparent homogeneity. Both the effect of inhibitors and the template specificity were clearly different compared with the two a-like DNA polymerases purified from wheat (Castroviejo et al., 1979, 1982). However, an antibody raised against this tow molecular weight DNA polymerase cross-reacted with DNA polymerase CII (a-type) from wheat (Castroviejo, unpublished results). The effect of NEM shows that the beet and pea small DNA polymerases are only inhibited at concentrations higher than 1 mM and seem to be more resistant than a-like DNA polymerase from the same source. Animal DNA polymerase a is resistant up to 20 mM NEM. Pea /3-like DNA polymerase is strongly bound to chromatin, as is the case of some animal DNA polymerases/3, but no evidence has been obtained that this association has a functional role and it is not explained by an unspecific binding. A chromatin-bound polymerase has also been found in developing soybean (D'Alessandro et al., 1980).
The role of plant /3-like DNA polymerases in DNA repair has not been proved. However, results have been reported that the UV light-induced DNA-repair synthesis in protoplasts of Nieotiana sylvestris is resistant to aphidicolin and thus it is not performed by an a-like DNA polymerase (Sala et al., 1982).
DNA polymerase The discovery of reverse transcriptase, the RNA-dependent DNA polymerase of retrovirus (Temin and Baltimore, 1972), led to the search for a similar enzyme in uninfected cells. A DNA polymerase able to recognize very efficiently a poly rA-oligo dT template was found in human cultured cells (Fridlender et al., 1972). This enzyme, called DNA polymerase 3', was different from the already described animal DNA polymerases a and /3 but it was also different from reverse transcriptase in that it was not able to copy a natural RNA template which is the main property of the retroviral DNA polymerase. DNA polymerase ~, from animal cells is found in the nuclear and mitochondrial fractions. It is the only DNA polymerase involved in the replication of the animal organellar genome (Weissbach, 1981). A similar enzyme of 100 kd was characterized from wheat (Tarrago-Litvak et al., 1975). The wheat enzyme, called DNA polymerase A (Castroviejo et al., 1979), resembles animal DNA polymerase 3' in its exquisite efficiency with poly rA-oligo dT template, as well as by the KC1 stimulation, the strong inhibitory effect of ddTTP and ethidium bromide and the resistance to aphidicolin. Nevertheless, the wheat DNA polymerase ~, is not localized in plant mitochondria and its role has not been elucidated; it is interesting to point out that this enzyme is able to recognize, although to a low extent, a natural RNA template like turnip yellow mosaic virus RNA and avian myeloblastosis virus RNA (Litvak et al., 1984). Wheat DNA polymerase A (3,-like) is also able to initiate DNA synthesis from an RNA primer, a property which differentiates this enzyme from the two a-like polymerases from the same source. Moreover, this enzyme is found associated with a DNA primase activity described in wheat embryos (Graveline et al., 1984). A DNA polymerase able to use poly rA-oligo dT template has been found
87 in healthy turnip cells (Volovitch et al., 1984; Dunham and Bryant, 1986). The spinach chloroplastic DNA polymerase has also been described as a v-like enzyme (see below). A 3-kd protein factor able to inhibit specifically the activity of a DNA polymerase isolated from cauliflower inflorescence, when the template poly rA-oligo dT is used, has been reported (Chou et al., 1981).
Chloroplast DNA polymerase Extranuclear plant cell organelles, chloroplast and mitochondria, contain their own genetic information which is replicated, transcribed and translated inside the organelles. Chloroplast contains a circular DNA of 130-150 kbp (Bohnert et al., 1982). The mechanism of replication or the chloroplastic DNA has not been elucidated but some information, gathered by electron microscopy studies, suggests that the mechanism can be by D-loop displacement, very similar to that described in animal mitochondria; "the rolling circle" mechanism has been also evoked in the replication or chloroplast DNA (Tewari, 1979). Isolated purified chloroplasts are able to synthesize chloroplast DNA in the presence of the appropriate precursors and Mg 2+ (Zimmermann and Weissbach, 1982). Lack of effect of some inhibitors like ddTTP and ethidium bromide on DNA synthesis in whole chloroplasts may be due to the lack of transport of these inhibitors inside the chloroplasts, since the same agents proved to be very efficient when organdie DNA synthesis was followed in chloroplast extracts from maize and liverwort suspension cultures (Zimmermann and Weissbach, 1982; Tanaka et al., 1984). Petunia chloroplast DNA synthesis in organello, however, was shown to be inhibited by ethidium bromide and ddTTP, these results may be explained by the non-integrity of petunia chloroplasts (Overbeeke et al., 1984). Aphidicolin does not inhibit DNA synthesis in whole chloroplasts or organelle extracts in all plant systems studied up to now. Using the technique of molecular hybridization it was shown that the chloroplast genome is somewhat amplified during growth of tobacco cells in the light (Cannon et al., 1985). The first attempt to characterize a chloroplast DNA polymerase was performed a long time ago (Spencer and Whitfeld, 1969). These authors
studied DNA synthesis in purified chloroplasts and described the solubilization of a DNA polymerase able to accept exogenous DNA as template. More recently DNA polymerases from purified chloroplasts of two plants have been isolated and characterized. The enzyme from spinach organelles (Sala et al., 1980) has been classified as a v-like DNA polymerase given its high efficiency with poly rA-oligo dT as template, while activated DNA, a native DNA which is gapped with very low amounts of pancreatic DNAase I, is almost not recognized by this enzyme. The enzyme has a relative M.W. of 105 kd and its activity is dependent on the presence of KC1 and Mn 2+, it is inhibited by NEM and resistant to aphidicolin. Surprisingly, the DNA polymerase from pea chloroplasts (McKown and Tewari, 1984) does not recognize a poly rA-dT template but it is inhibited, as the spinach polymerase, by NEM, ethidium bromide and is resistant to aphidicolin. The pea polymerase has been purified to apparent homogeneity; it is a monomeric protein of about 87 kd. No nuclease activity was detected in the purified pea DNA polymerase. It is not clear if the difference in template recognition, observed in the two chloroplast DNA polymerases mentioned above, is due to intrinsic enzymatic differences, to the higher degree of purification of the pea polymerase or to a partial proteolysis of the latter enzyme which would explain the difference of about 20 kd in the molecular weight of both enzymes.
Mitochondrial DNA polymerase The mitochondrial (mt) genomes of animal and plants differ in many respects, although the physiological function of mitochondria from all sources seems to be the same. The differences include size, structure and complexity, gene structure and codon usage (Pring and Lonsdale, 1985; Bendich, 1985). A striking finding that exemplifies the complexity of the plant mt genome is the size range that can vary from 218 kbp in turnip to about 2500 in watermelon. Surprisingly the green alga Chlamydomonas has a mitochondrial genome of about 16 kbp. The main feature of higher plant mt is the presence of a master, circular chromosome and subgenomic circular molecules generated by recombination between directly repeated elements
88 of the genome (Mulligan and Walbot, 1986). The recent interest in the study of plant mt genome organization can be explained by the observation, 10 years ago, that alterations in the pattern of restriction fragments of mt DNA, but not chloroplast DNA, correlated with cytoplasmically inherited male sterility. It is interesting to point out in this respect the presence of plasmid-like DNAs in the mitochondria of some maize cytoplasmic male sterile strains. The genomic structure of these plasmid-like DNAs differs from that of the mt DNA since they are linear and have a covalently bound protein at their 5' end; thus, they resemble the structure of some viral genomes such as q~29 and adenovirus (Laughan and Gabay-Laughan, 1983). As in the case of chloroplasts, isolated plant mitochondria are able to synthesize mt DNA (Ricard et al., 1983; Bedinger and Walbot, 1986). The mitochondrial genome is extensively labelled although no direct evidence of DNA initiation is presented. DNA synthesis in wheat mitochondria is strongly inhibited by NEM, ethidium bromide and ddTTP and it is resistant to aphidicolin. ATP stimulates the incorporation of dNTP precursors into mt DNA in wheat organelles, while it inhibits DNA synthesis in maize mitochondria. Nitrofurantoin, a potent inhibitor of bacterial growth, has proved to decrease strongly DNA synthesis in mitochondria from 48 h etiolated Vigna sinensis seedlings (Palit et al., 1980). A mt lysate has been obtained from wheat mitochondria and the DNA synthesizing properties of the system have been described (Ricard et al., 1983). DNA synthesis involves the action of a DNA primase activity confined to the mt compartment which synthesizes the RNA primers used by the mt DNA polymerase to initiate organelle DNA synthesis (M. Echeverria, unpublished results). A DNA polymerase with an apparent native molecular weight of about 180 kd has been solubilized, purified and characterized from wheat mitochondria (Castroviejo et al., 1979; Christophe et al., 1981). The enzyme is clearly distinct from the other DNA polymerases described in wheat and also differs from the animal mt DNA polymerase in the inability to use, with manganese or magnesium ions, a poly rA-oligo dT template, which is the preferred template of the animal
enzyme. Thus, the plant mitochondrial DNA polymerase cannot be classified as a T-like polymerase. A similar enzyme has been found in the mitochondria of rice cells (F. Sala, unpublished results). However, the strong effect of inhibitors like ddTTP, ethidium bromide and NEM, as well as the resistance to aphidicolin, seem to be similar in mt DNA polymerases from all eukaryotic cells studied up to now. A preliminary report on the mt DNA polymerase from cauliflower inflorescence has also been published (Fukasawa and Chou, 1980a).
Plant viral DNA polymerases The caulimoviruses are the only group of plant viruses known to contain double stranded DNA. The type member of the group, cauliflower mosaic virus (CaMV), is by far the most studied (Hohn et al., 1982). In parallel with the studies aimed at using this virus as an eukaryote vector in plant genetic engineering, several groups became involved in the study of the expression of the CaMV genome. One of the biggest surprises of these last years in the domain of plant molecular biology was the discovery that CaMV, although a DNA virus, uses RNA as a replicative intermediate and employs a reverse transcriptase for genome replication (Pfeiffer and Hohn, 1983; Hull, 1984; Hohn et al., 1985). This situation, the involvement of a reverse transcriptase, is similar to that of the replication of hepatitis B virus which has also a DNA genome and of the retroviruses which have an RNA genome. It is generally accepted that the DNA polymerase which replicates CaMV is, at least in part, coded by gene V product (ORF V) of the viral genome. The determination of the whole sequence of CaMV DNA allowed the finding that significant homologies exist between the gene V product and reverse transcriptase from hepatitis B virus and retroviruses (Volovitch et al., 1984; Toh et al., 1984). Based also on the sequence of CamV DNA it has been proposed that a methioninespecific tRNA may be involved in the priming of the reverse transcriptase reaction, in a similar way to the mechanism of retroviruses replication. The enzymological characterization of the DNA polymerase involved in CamMV DNA replication has been hampered by the low level and instability of the enzyme, as well as by the background
89 activities of the host D N A polymerases. A polymerase activity, very active with poly rA and poly rC as templates, was observed in turnip infected leaves. The activity could be chromatographically separated from D N A polymerase 3' (Volovitch et al., 1984). Other authors have been unable to detect differences at the level of D N A polymerases in healthy and CaMV infected turnip (Dunham and Bryant, 1986). The presence of a reverse transcriptase activity has been observed in viral replication complexes obtained by hypotonic shocks of viral infected organelles. CaMV D N A synthesis is insensitive to aphidicolin but partially inhibited by ddTTP (Pfeiffer et al., 1984). The same authors used the "activity gel" technique to characterize the CaMV D N A polymerase after polyacrylarnide gel electrophoresis in the presence of sodium dodecyl sulphate and protein renaturation (Spanos and Hubscher, 1982). The enzyme involved in viral replication coincided with a band of 75 kd, while an aphidicolin-sensitive D N A polymerase, present in healthy and infected leaves, was shown at the level of a l l 0 - k d peptide. A low but significant D N A polymerase activity has been detected within the CaMV particle; the molecular weight of this polymerase determined by activity gel is 76 kd (Menissier et al., 1984). The D N A polymerase activity associated to CaMV-replicating complexes was specifically inhibited by immune serum raised against a synthetic peptide corresponding to a portion of the viral gene V (ORF V) protein product (Laquel et al., 1986). The gene coding for ORF V has been cloned and expressed in the yeast Saccharomyces cerevisiae. Yeast expressing this gene accumulated significant levels of reverse transcriptase activity (Takatsuji et al., 1986). These and the previous results suggest strongly that CaMV code for a DNA polymerase different from the host DNA polymerases and able to act as a reverse transcriptase. Information on the replication of other plant D N A viruses is very scarce. Some results concerning the geminivirus have been presented (Coutts and Buck, 1985). The geminiviruses are a group of plant viruses whose members are characterized by the possession of twin isometric particles and genomes of single-stranded D N A circles between 2.5 and 2.7 kb. Nuclei and nuclear extracts from tomato golden mosaic virus (TGMV) infected
tobacco leaves were used for in vitro DNA synthesis. The synthesis of virus-specific D N A was resistant to aphidicolin, sensitive to N E M and ddTTP and stimulated by KC1 and ATP. T G M V is a two-component geminivirus (A and B). No component alone can be replicated when inoculated on tobacco leaves. In a recent study (Rogers et al., 1986) multimers of the A or the B component of T G M V were cloned separately in the T-arm of a disarmed Ti plasmid. Infection and regeneration of Petunia leaf discs led to no symptoms. When the plants containing the A or B components were crossed, however, plants showing T G M V infection symptoms were recovered with the expected frequency. Interestingly, plants with the A component alone (but not the B component) showed, by Southern blotting, replication of single- and double-stranded viral DNA. These results suggest strongly that viral DNA polymerase functions (the enzyme itself or a factor needed for a host polymerase) were encoded by the A component.
Acknowledgments Work performed in the authors laboratory was supported by C.N.R.S., the University of Bordeaux II and N.A.T,O. The authors are grateful to many colleagues for providing reprints and preprints of research articles and to Dr. L. Tarrago-Litvak for helpful comments on the manuscript.
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