Biotechnol Lett (2012) 34:205–212 DOI 10.1007/s10529-011-0764-8
ORIGINAL RESEARCH PAPER
Transient expression and activity of human DNA polymerase iota in loach embryos Irina V. Makarova • Andrey A. Kazakov • Alena V. Makarova • Nella V. Khaidarova • Larisa V. Kozikova • Valentina V. Nenasheva Leonid V. Gening • Vyacheslav Z. Tarantul • Ludmila E. Andreeva
•
Received: 18 July 2011 / Accepted: 29 September 2011 / Published online: 8 October 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Human DNA polymerase iota (Pol i) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol i preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol i activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol i activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol i. The overexpression of Pol i was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated
Electronic supplementary material The online version of this article (doi:10.1007/s10529-011-0764-8) contains supplementary material, which is available to authorized users. I. V. Makarova A. A. Kazakov A. V. Makarova (&) N. V. Khaidarova V. V. Nenasheva L. V. Gening V. Z. Tarantul L. E. Andreeva Department of Viral and Cellular Molecular Genetics, Institute of Molecular Genetics of Russian Academy of Sciences, 2 Kurchatov Sq, Moscow, Russia 123182 e-mail:
[email protected] L. V. Kozikova Russian Research Institute for Farm Animal Genetics and Breeding, St. Petersburg-Pushkin, 55-A Moskovskoe shosse, Tyarlevo, Russia 196625
Pol i expression may provide insights into Pol i functions in vertebrate animals. Keywords DNA polymerase iota Loach embryos Pycnotic nuclei Transient expression
Introduction Human DNA polymerase iota (Pol i) is a member of the Y-family of DNA polymerases that exhibits a number of unusual properties. Pol i may be responsible for maintaining genome stability as it participates in DNA translesion synthesis (Frank and Woodgate 2007; Washington et al. 2004) and base excision repair of oxidized DNA damages (Petta et al. 2008). Human Pol i also shows very low accuracy of synthesis when replicating undamaged DNA and displays striking template dependent differences in misincorporation frequencies. It exhibits high fidelity opposite template A but incorporates dGTP opposite T more efficiently than the correctly paired dATP (Frank and Woodgate 2007; Zhang et al. 2000) (so-called ‘‘misGvA – misincorporation of G versus A activity). The biological role of human Pol i ‘‘misGvA’’ misinsertion activity remains unknown. Pol i orthologs have been identified in many eukaryotes besides mammals: insects, fishes, amphibians and some fungi. Drosophila Pol i mainly incorporates correct dATP opposite T (Ishikawa et al. 2001) but little is known about characteristics of Pol i in other organisms.
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The majority of in vitro studies of replication properties of Pol i have been limited to analysis of DNA synthesis by purified recombinant protein. However, the in vivo activity of Pol i is likely regulated by post translational modifications and accessory factors. Indeed in human cells, Pol i is a part of a protein complex of *130 kDa (Sabbioneda et al. 2008). Therefore, the search for new methods and models for analysis of expression, biochemical properties, functions, and regulation of Pol i is urgent. As a step to understanding the in vivo functions of Pol i, we recently proposed a method of determination of Pol i activity in crude cell extracts of animal tissues and human tumors which was based on the detection of Pol i-specific ‘‘misGvA’’ activity (Gening et al. 2006; Kazakov et al. 2008). We further increased the sensitivity of the assay by replacing Mg2? ions with Mn2? cations and applied it for testing activity of human Pol i produced in yeast (Makarova et al. 2011). In the present work, we used the method of Pol i activity detection to monitor the transient expression of human Pol i during loach embryos development after the introducing of POLI gene into the fertilized loach eggs. Human Pol i was shown to be functionally active in loach embryos. We also tested the influence of Pol i expression on cell proliferation and viability during embryo development and observed an increase in the frequency of pycnotic nuclei in cells as well as some abnormalities in development of embryos expressing human Pol i.
4–6°C. Males and females were maintained separately. Mature eggs were obtained 40–42 h after an injection of 100 ME chorionic gonadotropin into a female and were fertilized by sperm solution supplement. Injections of linear DNA were performed into loach eggs under the blastodisc before the first division of the zygote using FemtoJet (Eppendorf) microinjector (320 hPa, 0.1 s, 14 l diam. needle). The concentration of the plasmids was optimized to achieve pronounced expression (without growth of toxicity in control animals) and corresponded 20 ng/ll in most experiments. Water without DNA, vectors pCI-neo and pEGFP without POLI gene were used for microinjections into the loach embryos as negative controls. Embryos were maintained in settling-vat tap water at room temperature. To test DNA polymerase activity and monitor expression of Pol i loach embryos were used in the period of time from 24 h post-fertilization (hpf) to 21 days post-fertilization (dpf). In total, seven independent experiments were carried out using about 2,600 embryos from seven parental couples. To test the viability of loach embryos and the frequency of abnormalities in development calculation of the percentages of survivor individuals and individuals with abnormalities was done on 4th and 9th dpf. The means and the standart errors of means (SEM) were calculated for three independent experiments using 1,300 embryos from three parental couples, each group of animals consisted of 100 embryos. Results were analyzed statistically using the Fisher’s exact test.
Materials and methods
Assay of DNA polymerase activity in cell extracts
Transient expression of human Pol i in embryos of loach Misgurnus fossilis L
Fresh cell extracts of the whole bodies of fish embryos (6–12 in the extract) were used as the enzymatic source for DNA-polymerase reaction with oligonucleotide substrate. The cell extracts were prepared as previously described (Gening et al. 2006; Kazakov et al. 2008). The three complementary oligonucleotides (the 17 nt universal primer and two 30 mer templates) were gel purified and used to test ‘‘misGvA’’ activity. The primer 50 -GGAAGAAGAAG TATGTT-30 and template 1 – 50 -CCTTCTTCATT CTAACATACTTCTTCTTCC-30 were the same as in the works (Gening et al. 2006; Zhang et al. 2000). The template 2 – 50 -CCTTCTTCATTGTAACATACTT CTTCTTCC-30 was described in work (Makarova et al. 2011). The template 2 is similar to template 1, but
Plasmid p6-1 encoding human Pol i was provided by Dr. R. Woodgate. The NcoI/BamHI-fragment of plasmid p6-1 together with a 60 nt part of the 50 untranslated sequence of POLI cDNA was cloned into plasmid pCI-neo (Promega) under the CMV promoter, resulting in plasmid pPINC. The NcoI/BamHI-fragment was also cloned into plasmid pEGFP-CI (Clontech) under the control of enhanced CMV promoter to obtain an in frame fusion of POLI with egfp at N-end of polymerase (plasmid pEGFP-POLI). Adult loaches were collected in the rivers of Russian middle region in December and kept at
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contains G instead of C at position 19 of the 30-mer template (underlined). For this template, primer misincorporation of G opposite T can not occur by ‘‘transient misalignment’’ mechanism. Oligonucleotide substrate was prepared by annealing of the (c-32P)-ATP 50 -labeled primer to the unlabeled template DNA at a molar ratio of 1:1.5. The standard reaction mixture (final volume 20 ll) contained 50 mM Tris–HCl (pH 8.0), 30 nM of the oligonucleotide substrate, 4 ll of tissue extract, 0.2 mM MnCl2, 0.5 mM of dATP and dGTP. The reaction was carried out at 37°C for 10 min. The reaction was terminated by cooling on ice followed by addition of an equal volume of a formamide loading dye. The replication products were separated in 20% polyacrylamide/8 M urea gel, visualized and quantified using a STORM 840 PhosphorImager (Amersham Biosciences, UK) and ImageQuant software. Western blot analysis Cell extracts of loach embryos prepared for the ‘‘misGvA’’ assay were used for western blot analysis. Samples containing 60 lg of total protein were subjected to electrophoresis in 8% SDS–PAGE gels, electro-transferred to PVDF membrane and probed with a 1:1,000 dilution of anti human Pol i polyclonal antibody (13635-1-AP, Proteintech Group Inc, USA). Proteins were visualized using the WesternBreeze Chromogenic Kit (Invitrogen). Morphological analysis of cell nuclei Loach embryos injected with pPINC plasmid, uninjected embryos and embryos injected with water or control plasmids were used for morphological analysis of cell nuclei at 96 hpf. pCI-neo, pEGFP and plasmid pCMVLacZ encoding E. coli b-galactosidase (Andreeva et al. 2008) were used as control plasmids. Ten morphologically normal embryos and 10 embryos with abnormalities in development were used for analysis in each group of loaches. For preparation of cytogenetic samples the whole fish embryos were incubated in 0.4% colchicine for 30 min in darkness and placed in solution of 1.1% Na citrate followed by removal of yolk sac. Then embryos were fixed in methanol–acetic acid (3:1) for 20 min, tissues were disrupted in 50% acetic acid and stained by the Romanovsky-Giemsa method. The total number of
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calculated cells was 1100–3500 for every 10 embryos. The results were analyzed statistically using the Mann–Whitney U test.
Results Transient expression of human Pol i in loach embryos Various fish species are widely used in mammalian gene transfer experiments for testing activity of heterologous proteins expressed under control of native or allogenic promoters. Loach embryos represents a convenient model for fast screening and studying of transient expression of transgenes due to simplicity of maintenance of adult individuals, embryos, and early fry (Andreeva et al. 2008). Embryos of loach, Misgurnus fossilis L. were used as a model for testing of human POLI gene expression. To ensure efficient expression of Pol i, we cloned the POLI cDNA gene under the control of the strong CMV promoter into plasmid pCI-neo, resulting in plasmid pPINC. To visually monitor expression we also produced the plasmid pEGFP-POLI (based on pEGFP-CI plasmid) encoding for human Pol i fused at the N-end with EGFP under the control of CMV promoter. Plasmids pPINC and pEGFP-POLI were injected into the loach eggs obtained after artificial fertilization. We confirmed the expression of recombinant Pol i protein in embryos by western blot analysis (Fig. 1a). It was found that human Pol i can only be detected in embryos which were injected with the pPINC and pEGFP-POLI plasmids with the POLI gene and not in control embryos injected with the pCI-neo and pEGFP vector. The expression levels of EGFP-fused Pol i and untagged protein were similar though EGFP-Pol i was found to be somewhat more stable to proteolysis compare to untagged Pol i (Fig. 1a). We then monitored the expression the POLI gene in loach embryos by detection of fluorescence of chimeric EGFP-Pol i protein and using ‘‘misGvA’’ activity assay. After injection of pEGFP plasmid encoding for EGFP alone at the concentration of 20 ng/ll or even 10 ng/ll, strong expression of EGFP took place in 95% of individual embryos at the middle-late blastula stage (24 hpf). The brightest mosaic fluorescence was observed in cells of blastoderm (Fig. 1b). Starting 48 hpf we observed gradual decay of the EGFP
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Fig. 1 Transient expression of human Pol i in loach embryos. a Detection of human Pol i in loach embryos by western blot. The whole body extracts from loach embryos injected with pPINC, pEGFP-POLI, pCI-neo or pEGFP plasmids were prepared at 3 dpf. b Fluorescence image of transiently
expression of EGFP in loach embryos. Loach embryos were injected with pEGFP or pEGFP-POLI plasmids at indicated concentrations. The bright fluorescence of EGFP alone is detectable starting from 24 hpf while only weak EGFP-Pol i fluorescence is detectable 24 hpf
fluorescence in embryo cells and increase of fluorescence in yolk syncytium (Fig. 1b). At later stages (since 12 dpf), the EGFP expression was detected in individual cells or cell clones located generally in epidermal and myofibril cells until 20 dpf (data not shown). A similar pattern of transient expression was observed for the chimeric egfp-POLI gene after injection of the pEGFP-POLI plasmid. However fluorescence of chimeric EGFP-Pol i was weaker in contrast to the EGFP protein when the same concentration of the vector (20 ng/ll) was used. The fluorescence of EGFP-Pol i protein was especially poor at the blastula stage and was observed at 24 hpf only when expression vector was injected at two-fold concentration (40 ng/ll) (Fig. 1b). The low EGFP-Pol i fluorescence signal may be explained by the interruption of appropriate EGFP molecule folding, distinctions in stability of EGFP and EGFP-Pol i proteins or regulation of their expression. Nevertheless EGFP-Pol i fluorescence was clearly detected starting 48–72 hpf during loach development with maximum at 4–7 dpf. About 15% of injected fish embryos did not reveal fluorescence and, therefore, did not produce the EGFP-Pol i protein (see below).
ability of Pol i to preferentially insert dGTP opposite T (‘‘misGvA’’ assay) (Gening et al. 2006; Kazakov et al. 2008; Makarova et al. 2011). The principle of the method is shown in Fig. 2a. The DNA polymerase reaction is carried out in the presence of cell extract, oligonucleotide substrate containing template T downstream of 17-mer primer and dATP and dGTP. 18-mer reaction products with A or G at the 30 -end are characterized by different electrophoretic mobility and Pol i activity can be detected by the presence of a typical double 18 nt band (upper G and lower A) after gel electrophoresis. Usually a replication stalling is observed at position 18 after dGTP-T misincorporation which facilitates detection of the corresponding reaction product. The analysis of Pol i activity by ‘‘misGvA’’ assay was prepared using the whole body extracts of loach embryos expressing Pol i, EGFP-Pol i or injected with control plasmids from 24 hpf to 21 dpf. The dynamics of ‘‘misGvA’’ activity in extracts of embryos expressing Pol i and EGFP-Pol i was very similar and generally coincided with the dynamics of EGFP-Pol i fluorescence. Prominent ‘‘misGvA’’ activity was revealed in loach embryos transiently expressing either Pol i or EGFP-Pol i from 2–3 to 15 dpf during development and the maximal activity of the enzyme was observed in 3–9 days-old fry in the most of experiments (Fig. 2b). No Pol i activity was detected after 15 dpf. No ‘‘misGvA’’ activity was detected in embryo extracts at any studied stage of development when
Analysis of ‘‘misGvA’’ activity in loach embryos transiently expressing human POLI Previously we proposed a method of Pol i biochemical activity assay in extracts of cells based on a unique
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Fig. 2 ‘‘misGvA’’ activity of human Pol i in loach embryos. a The scheme of the ‘‘misGvA’’ assay. DNA substrate contains T at position ?1 of the template downstream of 17-mer primer. DNA-polymerase reaction with cell extract is performed in the presence of only dATP and dGTP, allowing for the synthesis of DNA extension products up to 21 nucleotides. The higher mobility lower band of ‘‘G-T’’ mispair corresponds to the oligonucleotide with 30 -terminal A, whereas the upper band contains the less mobile oligonucleotide with a G at position 18.
b Dynamics of ‘‘misGvA’’ activity in loach embryos transiently expressing human Pol i. The DNA-polymerase activity in cell extracts of embryos injected with either pEGFP or pEGFP-POLI plasmids were analyzed on 1–16 dpf. Pol i protein expression level is shown by western blot (WB). c Analysis of the ‘‘misGvA’’ activity of the EGFP-Pol i protein in loach embryos injected with pEGFP-POLI plasmid that either reveal or do not reveal fluorescence signal (96 dpf)
water or control plasmids without POLI gene were injected into loach eggs (Fig. 2b). Importantly, embryos which were injected with pEGFP-POLI plasmid but did not show EGFP fluorescence did not demonstrate the ‘‘misGvA’’ activity (Fig. 2c). Thus, the appearance of ‘‘misGvA’’ activity of the fused EGFP-Pol i protein in embryo extracts was correlated with the fluorescence. The ‘‘misGvA’’ activity in extracts correlated with Pol i protein expression level as well (Fig. 2b). All experiments were also duplicated using DNA template 2 containing the substitution of nucleotide from C to G at 19-position to ensure that ‘‘misGvA’’ activity is not a result of dGTP incorporation by a template slippage mechanism (Makarova et al. 2011). The results for templates 2 were same as for
template 1 and are not shown. All together these data demonstrated that human Pol i transiently expressed in fish embryos after the plasmids injection is fully active and possesses specific ‘‘misGvA’’ activity. The influence of POLI expression on development of loach embryos The elevated levels of Pol i mRNA and protein (Albertella et al. 2005; Wang et al. 2007, 2010; Yang et al. 2004) as well as increased level of ‘‘misGvA’’ activity (Kazakov et al. 2008) were previously detected in several human tumors and tumor cell lines. Pol i was shown to cause the high frequency of UV-induced mutations in XPV cells (Wang et al.
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Table 1 The percentage of survived loach embryos (normal) and embryos with abnormalities in development on 4 and 9 dpf dpf
Embryos
Type of injection Uninjected
4
Survived With abnormalities
9
Survived With abnormalities
Water
pCI-neo
pPINC
pEGPF
90.3 ± 2.2
90 ± 1.3
79.5 ± 7.5
57.3 ± 11.1
79 ± 2.5
2.9 ± 1.2
1.5 ± 0.6
2 ± 0.8
32.4 ± 11.1
18.5 ± 1.5
87.8 ± 3.8
88.7 ± 0.4
78.5 ± 8.5
52 ± 14
71 ± 2
5.4 ± 1.7
1.9 ± 1.2
6.6 ± 2
31 ± 8
18 ± 1
Data are presented as means (%) ± SEM
2007) and breast cancer cell lines (Yang et al. 2004). To analyze the possible in vivo consequences of Pol i overexpression we compared viability and some morphological and cytogenetic characteristics of loach embryos injected with the pPINC plasmid or control plasmids. We observed statistically significant (P \ 0.01) increase of apparent morphological abnormalities in development in the group of loach embryos expressing human Pol i. Among them were disturbance of epiboly at the blastula stage, dysmorphogenesis, delay in hatching, fragmentation of yolk syncytium layer, trunk abnormalities, such as spinal lordosis and others malformation. We have to emphasize, that embryo abnormalities have been common both control and experimental groups. The percent of loach embryos with abnormalities in development on 4th and 9th dpf in the control groups of uninjected embryos and embryos injected with water or pCI-neo plasmid was 1.5–6.6%. At the same time, in the case of the pPINC vector encoding for Pol i more than 30% of injected embryos revealed certain abnormalities. The viability of embryos expressing human Pol i was somewhat reduced (P \ 0.01) in comparison to the control groups. The data on the frequencies of abnormalities and viability of loach embryos are summarized in Table 1. The levels of ‘‘misGvA’’ activity in transgenic embryos with developmental abnormalities were similar or slightly higher to the ‘‘misGvA’’ values of ‘‘normally developing’’ larvae expressing Pol i (Fig. s1). To test whether overexpression of Pol i may affect cell proliferation and cell viability we studied nuclei morphology, calculated the percent of mitotic divisions and pycnotic nuclei in ‘‘transgenic’’ and ‘‘nontransgenic’’ loach embryos at 96 hpf. The amount of cells on stage of mitotic division did not differ significantly in groups of larvae expressing and not expressing human Pol i (data not shown). However,
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the percent of pycnotic nuclei in embryos expressing Pol i was higher than in control groups of embryos injected with water, pCI-neo, pEGFP plasmids or vector pCMVLacZ. Increase of pycnotic nuclei in embryos expressing Pol i was statistically significant both in larvae characterized by morphological abnormalities in development and in normally growing fry (P \ 0.001) but was more prominent in the group of embryos with abnormalities (Fig. 3a, b).
Fig. 3 Pycnotic nuclei in loach embryos transiently expressing human Pol i. a The picture of normal (‘‘N’’) and pycnotic (‘‘P’’) nuclei in loach embryo injected with the pPINC plasmid is shown. The cytogenetic samples were prepared from the whole fish embryos at 96 hpf and stained by the Romanovsky-Giemsa method. b Percent of pycnotic nuclei in embryos expressing human Pol i. Data for embryos with normal development are presented in white, for embryos with abnormalities in development are shown in grey
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Discussion Human Pol i shows very low accuracy of synthesis when replicating undamaged DNA. The enzyme violates the Watson–Crick rules of base pairing and incorporates mainly dGTP opposite template T (Frank and Woodgate 2007; Zhang et al. 2000). Pol i functions are still unclear thus necessitating the search for new methods and models for studies of Pol i expression, biochemical properties, functions, and regulation. Previously we introduced the ‘‘misGvA’’ method which could be applied as a simple approach to monitor Pol i activity and replication fidelity in various living cells and tissues types (Gening et al. 2006; Kazakov et al. 2008; Makarova et al. 2011). The method does not require enzyme purification and allow detecting Pol i activity in the presence of accessory factors and possible posttranslational modifications. In the present work we developed a transient expression system for rapid analysis of human Pol i expression and activity using embryos of loach Misgurnus fossilis L. The prominent Pol i activity was detected in ‘‘transgenic’’ loach embryos using the ‘‘misGvA’’ method from 2 to 15 dpf during development with the maximal activity at 3–9 dpf. ‘‘misGvA’’ activity of EGFP-tagged Pol i correlated with EGFPfluorescence and was not found in nontransgenic loaches and loaches expressing EGFP at all studied stages of development. These data confirm the efficiency and specificity of the ‘‘misGvA’’ method. Various organisms expressing allogenic genes are commonly employed to explore the function of individual mammalian proteins including specialized DNA-polymerases (Plosky et al. 2008; Sobol et al. 2003; Yoo et al. 1994) and may provide additional important information on Pol i physiological significance. It was shown that production of human Pol i in yeast cells did not result in increase of mutagenesis (Makarova et al. 2011) though expression of human Pol i in a methyl methanesulfonate sensitive strain of yeast led to a moderate protective effect from this methylating agent (Plosky et al. 2008). Vertebrate animals overexpressing Pol i were not reported. The simplicity of obtaining transgenic individuals and similarity of biochemical processes of DNA synthesis and repair between fishes and mammals suggest that transgenic fish lines may appear a good model for study of Pol i functions in vertebrates. Our results with transient expression of Pol i in loach Misgurnus
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fossilis L. embryos demonstrate that transgenic fish embryos may be used as an in vivo model for human Pol i studies. We have shown that both untagged and EGFPtagged human Pol i can be efficiently expressed under the control of the CMV promoter in loach embryos. Transiently expressed heterological human Pol i was shown to be biochemically and likely functionally active in cells of loach embryos. Moreover, we have found that the activity of human Pol i in embryos expressing recombinant DNA polymerase was accompanied by decrease of viability of embryos, increase of developmental abnormalities and a number of cells with pycnotic nuclei in embryos. The data obtained may suggest that Pol i is involved in hypermutagenesis and/or cell apoptosis. Though it should be noted that defined model provides a strong expression of Pol i at early stages of development and our results may be considered as preliminary. In conclusion, future experiments with fish embryos with moderate but stable constitutive Pol i overexpression (for example, using transgenic zebrafish lines) may provide important insights into Pol i physiological functions in vertebrate animals. Acknowledgements We thank Dr. Rodger Woodgate (National Institutes of Health, Bethesda, USA) for providing plasmid encoding human Pol i and Dr. Andrey Kulbachinskiy (Institute of Molecular Genetics, Moscow, Russia) for the careful review of this paper. This study was supported by the Program of fundamental research of the Russian Academy of Sciences ‘‘Biodiversity’’, the Federal Targeted Program ‘‘Scientific and scientific-pedagogical personnel of the innovative Russia’’ 2009-2013 (state contract §G335) and Russian Foundation for Basic Research Program.
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