1 Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1
2 Great Lakes Forestry Centre, Sault Ste Marie, ON, Canada, P6A 2E5
Correspondence
Peter J. Krell
pkrell{at}uoguelph.ca
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ABSTRACT |
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MAIN TEXT |
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The full-length Cf114 (v-trex) ORF was amplified by PCR with forward primer 5'-ACTAGGATCCATGGGCCCAATTC-3' and reverse primer 5'-CAGTCTAGATTACATGGGATTAAC-3', containing BamHI/XbaI sites (Sigma), digested with BamHI and XbaI and inserted in-frame into the pFastBac HT donor vector (Invitrogen), downstream of the 6xHis tag, by T4 DNA ligase and the sequence was confirmed (Guelph Molecular Supercentre, University of Guelph, Canada). This construct was transformed into DH10Bac competent cells containing a bacmid with a mini-attTn7 target site and a helper plasmid. Subsequent recombinant baculovirus particles were obtained by transfection of recombinant bacmid DNA into Sf21 cells using Cellfectin reagent (Invitrogen). Exponentially growing Sf21 cells at a density of 0·5x106 cells ml1 were infected with recombinant baculovirus at an m.o.i. of 0·1, grown at 27 °C in complete Grace's medium and harvested at 24, 48, 72 and 96 h post-infection. Infected cells were lysed in lysis buffer [20 mM phosphate (pH 8·0), 300 mM NaCl, 1 % Nonidet P-40, 5 mM 2-mercaptoethanol] and soluble and insoluble cell proteins were fractionated by three freezethaw cycles and centrifugation (10 000 g, 30 min). Production of recombinant V-TREX was analysed by 15 % SDS-PAGE and identified by Western immunoblotting using a Penta-His HRP conjugate kit (Qiagen). The His-tagged V-TREX protein had an apparent molecular mass of 28 kDa, similar to the expected 30 kDa, and was detected in both soluble and insoluble fractions at 4896 h post-infection (Fig. 1a and b). About 50 % of the V-TREX was recovered as soluble protein by 72 h post-infection, whereas the amount of insoluble V-TREX protein increased above 50 % when cells were harvested at 96 h post-infection (Fig. 1a
).
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For biochemical characterization of V-TREX, we first used 5'- and 3'-DIG-labelled oligo(dT)30 (referred to as dT30) (Synthegen) as substrates for the nuclease activity assays (Kruchen & Rueger, 2003). Later, dT30 (Sigma) labelled with 32P at the 5' or 3' end, generated by standard methods with [
-32P]ATP or [
-32P]dATP (PerkinElmer), respectively, was used. The 32P-end-labelled substrates were purified by using Sephadex G-25 mini quick spin oligo columns (Roche) and exonucleolytic activities were assayed by using both DIG- and 32P-labelled oligonucleotides in parallel.
To determine whether the affinity-purified, His-tagged V-TREX had nuclease activity, standard nuclease reactions (50 µl) containing 25 mM Tris/HCl (pH 9·5), 5 mM MgCl2, 2 mM DTT, 0·1 mg BSA ml1, 10 ng purified V-TREX µl1 and 0·8 ng 5'- or 3'-DIG-labelled dT30 µl1 were set up. For radioisotope-labelled substrates, 5 ng V-TREX µl1 and 12 nM 5'- or 3'-32P-labelled dT30 were used. Reactions were done at 30 °C and 5 µl aliquots were withdrawn at various times (Fig. 2ad). Reactions were stopped with 3·5 µl 6x loading dye containing 95 % formamide and boiled for 3 min. Samples were analysed on a 20 % denaturing acrylamide gel with 7 M urea, followed by autoradiography for the 32P-labelled samples and by dry-blot transfer onto a nylon membrane and detection by antibody and chemiluminescence substrate of the DIG-labelled bands. The laddering of bands in Fig. 2(a and c)
is suggestive of a 3'-excision activity. By using 3'-labelled dT30, only full-length dT30 was observed and that decreased with time during the assay (Fig. 2b and d
). This suggested that the enzyme cleaved from the 3' end and, moreover, did not exhibit 5'-exonuclease activity. Unlike the alkaline exonuclease of herpes simplex virus type 1, V-TREX did not also have endonuclease activities (Hoffmann & Cheng, 1979
; Bronstein & Weber, 1996
; Mikhailov et al., 2004
). The 3'-excision rate of V-TREX was much faster with the 5'-32P-labelled dT30 (Fig. 2c
) than with the 5'-DIG-labelled one (Fig. 2a
). Moreover, the level of digestion using 3'-DIG-dT30 was lower than that using 5'-DIG-dT30. Even digestion using the 5'-32P-labelled substrate was lower than has been reported for the mammalian TREX proteins (Mazur & Perrino, 2001
) and phage T4 DNA polymerase (Promega), which has 3'5' exonucleolytic activity in a pH 7·5 buffer. Complete degradation of dT30 was obtained with 0·02 U T4 DNA polymerase µl1 after 5 min incubation at 30 °C with either 5'-DIG- or 5'-32P-labelled substrates (data not shown). Taken together, these results indicated that this slower excision rate of V-TREX could not be attributed only to the presence of the 3'- or 5'-end DIG. For example, the preferred substrate for V-TREX may be mispaired 3' termini, as for the mammalian TREX proteins (Mazur & Perrino, 2001
), rather than dT30. Furthermore, our reaction conditions for V-TREX might not be optimal and/or viral or cellular cofactor(s) may also be needed for maximum function.
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The gradual digestion of the 5'-labelled substrate with time (Fig. 2a and c) indicated that V-TREX either degrades oligonucleotides in a processive manner, but only very slowly, or it acts in a distributive fashion, where dissociation and reassociation of the enzyme with substrate are the limiting factors. To evaluate this, substrate-competition experiments were performed with unlabelled oligonucleotide substrate as a competitor to trap any dissociated V-TREX. 5'-32P-labelled dT30 (12 nM) and purified V-TREX protein (5 ng µl1) were pre-incubated for 15 min at 4 °C in the absence of MgCl2. The reaction was initiated by addition of 5 mM MgCl2 with or without unlabelled dT30 (1·2 µM) and incubated at 30 °C for the times indicated (Fig. 2g
). Addition of 100-fold excess cold dT30 retarded the degradation of the labelled substrate, compared to the reaction without the competitor (Fig. 2c
). This indicated that V-TREX might dissociate from the oligonucleotide substrate before completing digestion, suggesting that V-TREX acts in a distributive manner.
V-TREX activity under different enzyme concentrations was also investigated under standard assay conditions for 60 min. Limited cleavage of the 5'-DIG-labelled dT30 occurred with 10 ng V-TREX µl1 and was almost complete with 20 ng µl1 (Fig. 3a), whereas digestion of 5'-32P-labelled substrate started at 1 ng V-TREX µl1 (Fig. 3b
). Increased amounts of V-TREX enhanced the degradation of the 5'-labelled substrate in both cases (Fig. 3a and b
). The lower activity with DIG-labelled dT30 may have been due to the terminal DIG label, which might affect enzymesubstrate interactions, as discussed above. Consequently, the terminal DIG-labelling system does not seem to be suitable for analysis of the polarity and kinetics of V-TREX function (Fig. 2a and b
). Nevertheless, DIG-labelled dT30 is more stable and convenient than 32P-labelled dT30 and was still valid for studying the enzymic activity. V-TREX activity increased at alkaline pH with an optimum of 9·5, dropping dramatically at pH 10 (Fig. 3c
). Furthermore, Mg2+ was required, with maximal activity at 5 mM and an inhibitory effect by 50 mM (Fig. 3d
). NaCl was not required for V-TREX activity and concentrations of 5 mM NaCl or more were inhibitory, with no activity at 150 mM (Fig. 3e
). Addition of as little as 0·01 mg BSA ml1 effectively stabilized V-TREX activity, whereas increases in V-TREX activity were not significant with higher amounts of BSA (Fig. 3f
).
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ACKNOWLEDGEMENTS |
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Received 8 September 2004;
accepted 14 September 2004.