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1 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980–8578, Japan
2 Genetic Dynamics Research Unit Laboratory, RIKEN Research Institute, Wako, Saitama, 351–0198, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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mutation suppresses the temperature sensitivity of the polymerase 
mutants hys2-1 and cdc2-1 as well as the synthetic lethality of cdc2-1 pol32 mutants, suggesting a role for Rad18 in modulating DNA replication. As Rad18 mediates ubiquitination of PCNA, we examined whether PCNA modifications affected its function in replication. Multicopy PCNA alleviated the replication defects of rfc5-1 strains, but not those of pol mutants. In contrast, multicopy PCNA-K164R had reduced ability to suppress the replication defects of rfc5-1, but alleviated those of pol mutants. The roles of sumoylated and ubiquitinated PCNA in rfc5-1 and hys2-1 mutants were addressed by using mutant backgrounds that selectively affected sumoylation (siz1), ubiquitination (rad18), polyubiquitination (rad5, mms2), or the ability of cells to perform translesion synthesis (pol
, pol
). Our results are consistent with the idea that the Rad18/Rad5/Mms2 polyubiquitination pathway is important for replication completion, perhaps by promoting a template switch type of DNA synthesis. |
Introduction |
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, encoded by RAD30 (Johnson et al. 1999), and polymerase , encoded by REV3 and REV7 (Nelson et al. 1996). In the error-free damage bypass mechanism, the Rad6–Rad18 complex is thought to cooperate with another ubiquitin-conjugating dimer, Ubc13-Mms2 (Hofmann & Pickart 1999), via a RING finger protein, Rad5 (Ulrich & Jentsch 2000), in the conjugation of lysine 63 (K63)-linked multiubiquitin chains to target proteins. However, the only ubiquitination target relevant to DNA metabolism identified so far is PCNA (Hoege et al. 2002), a processivity factor for DNA polymerases. PCNA is mono-ubiquitinated at K164 by Rad6 and Rad18, and then the Rad6–Rad18 complex cooperates with Rad5 and the Ubc13-Mms2 dimer to attach mono-ubiquitin chains to PCNA (Broomfield et al. 1998; Hofmann & Pickart 1999; Ulrich & Jentsch 2000; Hoege et al. 2002). In addition, PCNA is also modified with SUMO by Ubc9 and the SUMO-specific ligase, Siz1. PCNA sumoylation occurs primarily at K164, but also at K127, and when the Lys-164 residue of PCNA is changed to an arginine residue, the sumoylation at K127 is stimulated (Hoege et al. 2002). Polyubiquitination of PCNA was shown to be elementary for post-replicative DNA repair (Hoege et al. 2002; Haracska et al. 2004). Furthermore, mono-ubiqutination of PCNA is required for damage-induced mutagenesis and both sumoylation and mono-ubiquitination of PCNA were shown to contribute to spontaneous mutagenesis (Stelter & Ulrich 2003; Haracska et al. 2004).
We have previously found genetical interactions between Rad18, Mms2 and the second subunit of DNA polymerase , Pol31, which have suggested a regulatory role for Rad18/Mms2 in DNA polymerase -mediated replication (Branzei et al. 2002a). In this study, we extended our investigation on the functional interaction between Rad18 and DNA polymerase subunits. In addition, we investigated whether the Rad18 modulatory function on replication is linked to its ability to ubiquitinate PCNA and we examined the roles of PCNA modifications on replication in replication mutants. Our results suggest that polyubiquitination of PCNA is firmly linked to the ability of cells to complete replication, while the roles of mono-ubiquitination and sumoylation are secondary to those of polyubiquitination from the perspective of completing replication during replication stress.
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Results |
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We previously found that rad18 and mms2 mutations suppress the temperature sensitivity of hys2-1 mutants (Branzei et al. 2002a). hys2-1 mutants carry a mutation in the POL31 gene encoding the second subunit of DNA polymerase and were shown to exhibit replication defects at high temperatures (Sugimoto et al. 1995).
In this study, we analysed the effect of the rad18 mutations on cdc2-1, a pol3 mutant in which the activity of DNA polymerase is reduced (Sitney et al. 1989) and the repair of single-stranded DNA gaps is strongly inhibited under restrictive conditions (Suszek et al. 1993). We found that the rad18 mutation suppressed the temperature sensitivity of cdc2-1 mutants (Fig. 1A). It is reported that cdc2-1 pol32 strains carrying a URA3-POL32 plasmid are unable to form colonies on plates containing 5-FOA, a drug that selects for cells that have lost the URA3 marker, thus indicating that cdc2-1 pol32 cells are lethal (Gerik et al. 1998). Our studies confirmed the reported lethality of cdc2-1 pol32 strains and found that cdc2-1 pol32
rad18 strains carrying a URA3–POL32 plasmid are viable on plates containing 5-FOA, thus indicating that the rad18 mutation suppresses the synthetic lethality of cdc2-1 pol32 strains (Fig. 1B). Interestingly, rad18 was in an epistatic relationship with cdc2-1 with regard to HU sensitivity (Fig. 1A). These functional interactions suggested that Rad18 and DNA polymerase cooperate in vivo to cope with replication stress.
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The fact that Rad18 functions in the ubiquitin conjugation pathway raises the possibility that the effect of Rad18 on replication is due to its ability to ubiquitinate replication proteins. PCNA has essential functions in replication and is one of the Rad6/Rad18-ubiquitination targets. To verify our hypothesis, we examined whether ubiquitination of PCNA affects its function during replication.
As PCNA is a target for both sumoylation and ubiquitination, and the main modification site K164 of PCNA is shared by both modifications, we initially examined whether mutations at PCNA's modification sites affect its function in replication (Fig. 2A). We introduced K127R, K164R or both K127R/K164R mutations in POL30 subcloned in a multicopy plasmid. For simplicity we will refer to the POL30 gene as PCNA in this study. Previous studies have demonstrated that the mutations K127R and K164R do not affect the stability of PCNA but affect the ability of PCNA to be modified with SUMO or ubiquitin (Hoege et al. 2002). In order to examine the effects of these mutated forms of PCNA on replication progression, we assayed the effect of wild-type PCNA versus the effect of PCNA-carrying mutations at K127 and K164 on the replication defect of several replication mutants. In wild-type cells, none of these forms of PCNA interfered with the ability of cells to grow at normal or high temperatures (Fig. 2B). However, multicopy PCNA carrying a mutation at K164 caused HU and MMS sensitivity in wild-type cells (Fig. 2B). None of the PCNA forms affected the intra-S checkpoint of wild-type cells (data not shown), suggesting that the checkpoint pathway is active in these cells. Thus, the observation that multicopy PCNA-K164R causes MMS and HU sensitivity in wild-type cells is likely to suggest that PCNA modification at K164 is important for cells to complete replication in response to replication stress. Because these PCNA constructs did not show a phenotype of their own or growth defects in cells grown at different temperatures, their effect on the ability of replication mutants to complete replication at restrictive temperatures was analysed.
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The rfc5-1 cells were previously shown to have a replication defect as well as a growth defect at high temperatures (Sugimoto et al. 1996). Although, multicopy PCNA was shown to suppress the growth defect of rfc5-1 cells at restrictive temperatures, its ability to suppress the replication defect of rfc5-1 cells at high temperatures was not analysed previously (Sugimoto et al. 1996). We used pulse-field gel electrophoresis (PFGE) to analyse the effect of PCNA on the replication defect of rfc5-1 cells. Wild-type and rfc5-1 cells carrying vector or multicopy PCNA were grown at 25 °C and then shifted to 38 °C. Aliquots containing identical cell numbers were taken and the chromosomal DNA was analysed by PFGE. Consistent with previous reports (Sugimoto et al. 1996), rfc5-1 cells expressing vector have a large fraction of chromosomes that are unable to enter the gel and remain within the well at restrictive temperatures (Fig. 3A). In contrast, rfc5-1 cells expressing multicopy PCNA showed a normal pattern of chromosome migration, indicating that the replication defect of rfc5-1 cells at high temperatures is alleviated by multicopy PCNA (Fig. 3A). Therefore, the effects of PCNA on the replication defect and growth defect of rfc5-1 mutants at restrictive temperatures are intertwined.
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Effects of PCNA mutants on the replication phenotypes of pol mutants
Given the functional interactions between Rad18 and polymerase , and the fact that PCNA is a component of the polymerase complex, we addressed how PCNA and PCNA mutants affect the phenotypes of cells with mutations in DNA polymerase , such as hys2-1 and pol32. PCNA overproduction did not affect the phenotypes of pol32 cells but, interestingly, multicopy PCNA-K164R suppressed the HU sensitivity of pol32 mutants (Fig. 3C). Similar to the pol32 case, PCNA-K164R suppressed the temperature sensitivity and HU sensitivity phenotypes of hys2-1 cells (Fig. 3D). Therefore, multicopy PCNA and the mutated forms of PCNA have different effects on pol and rfc5 mutants.
We examined the flow cytometry profiles of hys2-1 cells to address whether the effect of multicopy PCNA-K164R on the ability of hys2-1 mutants to cope with replication stress can also improve the ability of these cells to progress through the cell cycle. hys2-1 cells carrying vector, multicopy PCNA and PCNA-K164R were grown under permissive and restrictive conditions and samples were taken at time intervals for flow cytometric studies. Consistent with previous reports (Sugimoto et al. 1995) and the idea that faulty replication leads to checkpoint activation and cell cycle arrest, hys2-1 mutants had a large population of cells in the G2/M phase of the cell cycle, at both permissive and non-permissive temperatures (Fig. 4A), and a large population of cells were present as large-budded cells typical of cells in the G2 and M phases of the yeast cell cycle (data not shown). These results suggested that the bulk DNA is replicated in these cells and this inference was confirmed by PFGE (data not shown). However, the G2 phase arrest of hys2-1 cells suggested that DNA is not suitable for division in these cells.
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-factor at 25 °C and then released into fresh medium at either 25 °C or 37 °C. hys2-1 cells tended to arrest in the G2/M phase of the cell cycle and this phenotype was aggravated at 37 °C (Fig. 4B). In contrast, hys2-1 cells expressing PCNA-K164R had a larger population of cells that completed mitosis and entered a new G1 phase as compared with the corresponding hys2-1 cells carrying vector or PCNA, although still smaller than the corresponding wild-type cells (Fig. 4B).
Although the effects of multicopy PCNA and PCNA-K164R were different in pol mutants as compared with rfc5-1 mutants, the results clearly suggested that modification of PCNA at K164 plays an important role during replication. In order to gain insight about the type of modification responsible for this function of PCNA during replication, we used additional mutant backgrounds that selectively inhibited sumoylation or ubiquitination of PCNA.
Inhibition of PCNA sumoylation does not affect its function in replication
SUMO modification of PCNA by Ubc9 and the SUMO-specific ligase Siz1 occurs primarily at K164, but also at K127 (Hoege et al. 2002). We analysed the effect of non-sumoylated PCNA in replication mutants, by examining the effect of PCNA in replication mutants that additionally carried a siz1 mutation. Multicopy PCNA could suppress the growth defect of rfc5-1 siz1 cells to the same extent as it did in rfc5-1 cells, suggesting that sumoylation of PCNA is not required for its ability to overcome the replication defects of rfc5-1 cells (Fig. 5A). As expected, the K164R mutation diminished the ability of multicopy PCNA to suppress the replication defects of rfc5-1 siz1 cells (Fig. 5A). Similar results were obtained with hys2-1 mutants; that is, the siz1 mutation did not significantly affect the effect of PCNA or PCNA-K164R in hys2-1 mutants (Fig. 5B). It is important to note that the growth of siz1 single mutants at high temperatures was not affected in any way by multicopy PCNA (Fig. 5A,B). These results suggested that the effect of PCNA and PCNA-K164R on the ability of rfc5-1 and hys2-1 cells, respectively, to complete replication was not due to its sumoylation.
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To assess the effect of PCNA ubiquitination on the ability of replication mutants to complete replication, we used a rad18 background in which ubiquitination of PCNA is abolished while sumoylation is not affected (Hoege et al. 2002). In an rfc5-1 rad18 strain, multicopy PCNA could only partially suppress the replication defect of rfc5-1 cells (Fig. 6A). It is important to note that wild-type or rad18 single mutant cells carrying vector, PCNA or mutated forms of PCNA did not have any difficulties in growing at high temperatures (Fig. 6C). These results suggest that the RAD18-ubiquitination pathway is essential for the ability of multicopy PCNA to suppress the replication defect of rfc5-1 cells.
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mutation suppresses the temperature sensitivity of hys2-1 cells [(Branzei et al. 2002a) and Fig. 6B]. This is reminiscent to the effect of PCNA-K164R on hys2-1 cells (Fig. 6B). Therefore, these results suggest that inhibition of PCNA ubiquitination promotes growth of hys2-1 cells at restrictive temperatures. The effect of PCNA on the replication mutants depends on its polyubiquitination by Rad18/Rad5/Mms2 pathway rather than on its ability to promote translesion synthesis
Next, we asked which functions of the RAD18 gene and which branches of the damage tolerance pathway affect the interaction between rfc5-1, hys2-1 and PCNA. Therefore, we analysed the effect of multicopy PCNA, PCNA-K164R, PCNA-K164R, K127R and PCNA-K127R in rfc5-1 cells when other genes required for error-free PRR and polyubiquitination of PCNA, such as MMS2 and RAD5, or genes required for translesion synthesis, such as REV7 or RAD30, were deleted. Multicopy PCNA retained its ability to suppress the replication defect of rfc5-1 when RAD30 or REV7 genes were deleted, but not when genes involved in the polyubiquitination of PCNA, such as MMS2 and RAD5, were absent (Fig. 6A). It is important to note that rad18, rad5, mms2, rad30 or rev7 mutants carrying multiple copies of PCNA did not have a growth defect at high temperatures (Fig. 6C).
Recently, it was reported that mono-ubiquitination and sumoylation of PCNA are important for Pol- and Pol-mediated translesion synthesis (Stelter & Ulrich 2003). Our results can therefore be interpreted as suggesting that TLS does not play a major role in completion of replication in rfc5-1 mutants, while the Rad18/Rad5/Mms2-mediated polyubiquitination of PCNA is crucial for this process.
We also analysed the effect of different PRR genes in regard to the effect of multicopy PCNA on hys2-1 cells. Mutations in REV3 or RAD30 did not affect the phenotypes of hys2-1 cells (Fig. 6B), while mms2 suppressed the temperature sensitivity of hys2-1 cells, in a manner similar to that of rad18 and PCNA-K164R
[Fig. 6B and (Branzei et al. 2002a)].
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Discussion |
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This study and other previous reports have found functional interactions between Rad18 and DNA polymerase
[(Giot et al. 1997; Branzei et al. 2002a; Chanet & Heude 2003) and Fig. 1]. The results of this study support the idea that Rad18 is implicated in the completion of replication through its ability to ubiquitinate PCNA, and perhaps other replication proteins. This ability of Rad18 to modulate DNA replication is dependent on the activity of other ubiquitin-conjugating and -ligating enzymes such as Rad5 and Mms2 to modify PCNA (see Fig. 6), and on the availability of the replication proteins themselves.
While previous studies have linked PRR and ubiquitination to the ability of cells to repair DNA damage (Prakash 1981; Torres-Ramos et al. 1996, 1997, 2002; Haracska et al. 2004), our results have further indicated a role for multiubiquitinated PCNA during S-phase to modulate replication during replication stress. The genetical interactions we have found between Rad18, Mms2 and DNA polymerase
[Fig. 1 and (Branzei et al. 2002a)] support the idea that Rad18 and Pol functionally interact and modulate replication fork activity. In addition, the finding that the replication defect of rfc5-1 cells at high temperatures can be suppressed by overproducing PCNA, but only partly by overproducing PCNA-K164R, suggests that the K164 modification of PCNA is important for this function. Further experiments in mutant backgrounds that selectively inhibited sumoylation, ubiquitination, polyubiquitination or TLS, suggested that polyubiquitination of PCNA is pivotal in promoting replication completion in rfc5-1 cells (Figs 5A and 6A). While mono-ubiquitination of PCNA appears to promote TLS bypass of DNA lesions (Stelter & Ulrich 2003; Haracska et al. 2004), our results indicate that this pathway is not significant in reactivating stalled forks caused by replication stress (Fig. 6). Mono-ubiquitination of PCNA might be activated only when the Rad5/Mms2/Ubc13 complex is not available to bind the Rad6/Rad18 complex and promote polyubiquitination of PCNA. Alternatively, it is possible that the DNA or protein complexes formed at a stalled fork are not efficiently bypassed by TLS polymerases.
We like to propose a model in which Rad18 binds the ssDNA that forms at stalled replication forks (Bailly et al. 1994) and then the Rad18/Rad6 complex, together with Rad5 and the Mms2/Ubc13 complex (Ulrich & Jentsch 2000), act to polyubiquitinate PCNA. We envision that the polyubiquitin chains of PCNA will transiently destabilize the DNA polymerase complex, leading to its dissociation from the replication fork and then its re-assembly for DNA synthesis, but using the daughter strand of the sister duplex as template for DNA synthesis (see Fig. 7). This mechanism is thought to also be responsible for repair of UV-induced DNA damage and is known as template switch or copy choice type of damage bypass (Higgins et al. 1976; Haracska et al. 2004). The requirement of RAD18, RAD5 and MMS2 in this pathway (Prakash 1981; Torres-Ramos et al. 2002) is perhaps due to their ability to poly-ubiquitinate replication proteins such as PCNA. Consistent with this idea, a very recent study shows that the pol30-119 mutation, which results from a change of the lysine 164 residue of PCNA to arginine, impairs the efficiency of post-replicational repair of discontinuities that form in the DNA synthesized from UV-damaged DNA templates (Haracska et al. 2004).
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subunits that affect its ability for DNA replication and/or DNA repair synthesis (see Torres-Ramos et al. 1996, 1997). It is likely that most pol mutants showing growth defects have unstable Pol complexes that cause increased fork stall and ultimately increased requirement for fork recovery. In addition, some mutations in Pol might dramatically affect the stability of the PCNA-Pol complex, and therefore activation of a template switch DNA synthesis mechanism in these cells is expected to be detrimental for growth and viability. Consistent with this idea, we observed that over-expression of PCNA slightly aggravated the replication defects of hys2-1 mutants as measured by HU sensitivity and temperature sensitivity (Fig. 3D), and multicopy PCNA-K164R suppressed the temperature sensitivity of hys2-1 mutants and the HU sensitivity of hys2-1 and pol32 mutants (Fig. 3C,D), linking this modification of PCNA at K164 with instability of the polymerase replication complex. This model accounts for the observations that both inhibition of RAD18/MMS2 function and inhibition of PCNA modification at K164 had similar effects on the ability of polymerase mutants to complete replication and that both the RAD18/RAD5/MMS2 ubiquitination pathway and PCNA modification at K164 were elementary for the ability of PCNA to suppress the growth/replication defects of rfc5-1 cells at restrictive temperatures (Fig. 6). Previous reports have found increased SUMO modification of PCNA in the S-phase and therefore suggested a role for PCNA sumoylation in replication (Hoege et al. 2002). The results of this study suggested that PCNA sumoylation does not play a major role in promoting completion of replication in replication mutants. However, as both SUMO modification and ubiquitination target the same residue, K164, of PCNA, it is reasonable to assume that sumoylation of this residue blocks ubiquitination and perhaps functions to direct the substrate for other functions. Taking into consideration the observations that UBC9 and SUMO interact with many proteins involved in recombination, and ubc9 mutants are defective in damage-induced recombination (Maeda et al. 2004), our speculation is that, in addition to its function in promoting mutagenesis, SUMO modification might promote restoring of broken forks by recombinational repair. We therefore envisage that SUMO modification and ubiquitination of substrates adequately modulate the restoration or repair of stalled or broken replication forks that are produced in eukaryotic cells during replication. Further research aimed at identifying new ubiquitin/SUMO substrates with functions in DNA replication and at elucidating how these modifications affect progression and completion of DNA replication, will perhaps help to elucidate the significance of these modification mechanisms in regulating S-phase progression and maintaining genome integrity.
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Experimental procedures |
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The yeast strains used in this study were constructed using standard genetic methods and are shown in Table 1. SIZ1, RAD18, MMS2, RAD5, REV7 and RAD30 were disrupted with the hygromycin resistant cassette according to the protocol described in (Goldstein & McCusker 1999; Branzei et al. 2002a). REV3 was disrupted by PCR by amplifying the rev3::LEU2 cassette from the strain yMP10382 which was a kind gift from Dr L. H. Hartwell and was described in (Paulovich et al. 1998).
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YEp195-POL30 was a kind gift from Dr K. Sugimoto and was described in (Sugimoto et al. 1996). Mutations K127R and K164R were introduced in POL30 by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) to obtain YEp195-POL30-K127R, YEp195-POL30-K164R and YEp195-POL30-K127R, K164R. The mutations were confirmed by DNA sequencing.
Analysis of drug sensitivity and growth ability by drop assay
Log-phase grown cells were harvested, washed once in distilled water, counted and diluted. Tenfold serial dilutions of cells (105, 104, 103 and 102 cells) were spotted on to YPAD or SC-Ura plates containing MMS or HU at the indicated concentrations. The plates were incubated at the indicated temperatures for 2–4 days and the cells were photographed.
Cell cycle analysis
Cell cycle analysis was performed as previously described (Branzei et al. 2002b). For synchronization experiments, cells were treated with -factor at a final concentration of 5 µg/mL for 3 h. After microscopic confirmation of G1 arrest, the -factor was washed away and cells were re-suspended in fresh YPAD medium. Samples were taken at time intervals and the yeast cells were fixed overnight at 4 °C in 1 mL of 70% ethanol. The fixed cells were washed, sonicated and re-suspended in 400 µL of 50 mM sodium citrate (pH 7.0) and then treated with ribonuclease A (0.25 mg/mL) for 1 h at 37 °C, followed by treatment with proteinase K (0.5 mg/mL) for 1 h at 50 °C. Propidium iodide was then added to a final concentration of 16 µg/mL, and cells were incubated for at least 12 h at 4 °C. The DNA content of cells was analysed by a Becton-Dickinson FACScan flow cytometer using the CellQuest system.
Pulse-field gel electrophoresis (PFGE)
For PFGE analysis of chromosomes, the indicated strains were grown overnight in selective media and then shifted to YPAD for 3–4 h until log phase at permissive temperatures. The log-phase cultures were appropriately diluted and then shifted to the indicated temperatures. At the indicated times, samples of cells were taken and agarose plugs of chromosomal DNA were prepared as described (Hiraoka et al. 2000). Electrophoresis was performed on 1% PFGE-certified agarose (Bio-Rad, Hercules, CA) in 0.5 x TBE buffer at 14 °C with a CHEF Pulsed-Field Electrophoresis-System (Bio-Rad) as previously described (Hiraoka et al. 2000).
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: branzei{at}postman.riken.go.jp
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Received: 25 May 2004
Accepted: 5 August 2004
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