- Email: [email protected]
Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells
Mutation Research 459 Ž2000. 65–71 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres
Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells Olga G. Shcherbakova a , Vladislav A. Lanzov a , Hideyuki Ogawa b, Michael V. Filatov a,) a
Molecular and Radiation Biophysics DiÕision, Petersburg Nuclear Physics Institute, Gatchina 188350, Russian Federation b Department of Biology, Osaka UniÕersity, Osaka, Japan Received 18 May 1999; received in revised form 14 October 1999; accepted 22 October 1999
Abstract The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells. q 2000 Elsevier Science B.V. All rights reserved. Keywords: RecA protein; Gene targeting; Homologous recombination in somatic mammalian cells
1. Introduction Gene targeting in mouse embryonic stem ŽES. cells, if successful, provides a powerful method for systemic alteration of mammalian genome as well as for studying gene functions in vivo w1x. Different experimental strategies for targeted gene modification in the desired chromosomal loci of ES cells via
) Corresponding author. Tel.: q7-81271-46626; fax: q781271-32303; e-mail: [email protected]
homologous recombination have been developed recently. The major difficulty in general application of these strategies is the fact that homologous recombination in ES cells is an infrequent event w2,3x. Several factors — the length of homologous fragment in input DNA w5x, the presence of double strand breaks in the targeted genomic locus w4x, the usage of targeting vectors bearing DNA isogenic to DNA of the host cells w5x — are influenced much on frequency of homologous targeted events. The introduction of an active exogenous recombinase is one of the possible approaches to increase the
0921-8777r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 5 9 - 2
66
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
efficiency of the gene targeting in mammalian cells. The Escherichia coli RecA protein has been shown to be responsible for two main steps of recombination process: the search for homology and for strand exchange between two DNA molecules w6x. A number of proteins with structural and functional similarity to RecA have been found in three major domains of life — Prokarya, Archaea and Eukarya w7x. A human HsRad51 protein has been shown to possess both DNA-dependent ATPase activity and capability to form a nucleoprotein filament that resembles that of RecA w8x. Although the rates of ATP hydrolysis, homologous pairing and subsequent strand exchange promoted by the HsRad51 appear to be less efficient, the functional and structural similarity of HsRad51 and RecA becomes obvious. These facts prompted us to study the possibility of whether the additional production of bacterial RecA protein in mammalian cells can specifically increase the frequency of homologous recombination. For this aim, the E. coli RecA protein fused with a nuclear localization signal ŽNLS. was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of targeted inactivation of hprt gene was 10-fold increased in the mouse cells expressing the RecA protein.
2. Materials and methods 2.1. Plasmids The pATR4 plasmid was a kind gift of Dr. K. Kohno ŽInstitute for Molecular and Cellular Biology, Osaka, Japan.. The plasmid contains the recA gene fused with the NLS of the SV40 large T antigen ŽPro–Lys–Lys–Lys–Arg–Lys–Val–Glu–Asp–Pro. that specifies the nuclear translocation of the SV40 large T antigen w16x. The expression of the recA gene in mammalian cells was provided by replacement of original recA promoter with chicken b-actin promoter and Kozak’s sequence in pATR4 plasmid w9x. This plasmid also bears the selection marker, hph Žhygromycin B phosphotransferase. w9x. The pVR9.1 plasmid, kindly provided by Drs. K. Thomas and M. Capecchi ŽUniversity of Utah, USA.,
contains the 9.1-kb fragment of hprt gene with insertion of the selectable marker, neo gene w5,10,11x. In order to generate targeting vector, the pVR9.1 plasmid was cleaved by BglII restriction endonuclease. 2.2. Cell culture, transfection, and selection of drug-resistant cells Mouse embryonic teratocarcinoma cells, line F9, were grown in L-glutamine containing Eagle medium ŽBiolot, Russia. supplemented with 10% bovine serum ŽBiolot, Russia. and gentamycin sulphate ŽSigma.. DNA was introduced into the F9 cells by electroporation. Prior electroporation growing cells were trypsinised, washed with Eagle medium and counted. In each experiment, 1.8 mg of DNA ŽpRV9.1, linearized by BglII. has been used per 1 = 10 6 cells taken for electroporation. Cells were mixed with DNA and electroporated at 1.08 kV and 0.2 mF. Under these conditions, 50 " 10% of the cells survived the procedure. Then cells were placed in prewarmed complete culture medium and 48 h later cells were trypsinised and counted. The 2–4 = 10 5 cells per flask were plated in the complete medium supplemented with 500 mgrml G418 ŽSigma.. The cells were maintained under G418 selection for 9–11 days, until G418 R colonies have become visible. The colonies in control flasks were fixed and counted in each experiment. After selection with G418, the growth medium was supplemented with 0.2 mgrml 6-thioguanine Ž6-TG; Sigma. and cells were maintained about 10 days of the selection. At the end of this period, the number of colonies, doubly resistant to G418 and 6-TG, was determined. 2.3. Southern blot analysis F9 cell lines resistant to G418 and 6-TG were grown under the standard condition. Genomic DNA was purified from the cells using a standard procedure w12x. These DNAs were digested with EcoRI or XbaI enzyme and separated on 0.8% agarose gel. DNA was transferred to Hybond-N ŽAmersham. nylon membrane by alkali transfer protocol w12x, prehybridised with 6 = SSPE, 0.2% SDS, 10 mgrml
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
heparin ŽSpofa., and hybridised in the same buffer for 17 h at 658C with probes a or b w13x. Probe a is a 32 P-labeled single-stranded fragment of DNA, obtained by one-primer polymerase chain reaction ŽPCR. amplification with the primer. 5X-TCGAGAGGTCCTTTTCACC used 800 bp EcoRI–BstEII fragment of pRV9.1 vector containing part of exon 7 of hprt gene as the template. Probe b was a 32 P-labeled single-stranded fragment of DNA obtained in PCR reaction with the primer, 5X-TCCTGTTGGATGTTGCCAGTAAA using the 1000bp EcoRI fragment of pRV9.1 vector containing part of exon 9 of hprt gene as the template. Probes a and b were used for hybridization with EcoRI- and XbaI-digested genomic DNAs correspondingly. 32 P signals were revealed by autoradiography with the enhancing screen at y708C. 2.4. Production of the cell lines expressing T-RecA protein DNA of pATR4 plasmid was electroporated into F9 cells by the procedure described above. Fortyeight hours after electroporation, cells were plated in Eagle medium, supplemented with 10% bovine serum and 400 mgrml Hygromycin B ŽSigma.. After 10 days of the selection, colonies resistant to the Hygromycin B, and therefore harboring pATR4 plasmid in their genomes, were picked up and grown under the standard conditions. When cells reached confluence, they were trypsinised, harvested and resuspended in Laemmli w14x sample buffer without bromophenol blue. After heat denaturation, lysates were clarified by centrifugation and protein concentration was determined by Bradford w15x method. About 20–40 mg of protein per line was subjected to 12.5% polyacrylamide SDS gels w14x. Resolved proteins were transferred to nitrocellulose membranes by the standard protocol w12x. RecA protein was revealed using the polyclonal rabbit anti-RecA antibodies in concentration 0.1 mgrml in PBS-0.05% Tween-20 buffer with 5% non-fat dry milk as blocking reagent. The first antibody against RecA protein was raised in rabbits by repeated intradermal injections of purified RecA protein with Freind’s adjuvant ŽCalbiochem, Behring, La Jolla.. Antigen–antibodies complexes were visualized by peroxidase-conjugated swine anti rabbit immunoglobulins ŽDako..
67
3. Results 3.1. Generation of mouse cell line expressing nucleus-targeted RecA protein In order to investigate the influence of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, pATR4 plasmid containing the E. coli RecA protein encoding gene fused to NLS from large T antigen of SV40, described above, was introduced into the mouse F9 teratocarcinoma cells. The RecA protein modified by fusion with the translocation signal ŽT-RecA. has been shown to keep the original activity of the RecA protein and to be efficiently transported into the nuclei of mammalian cells w9,17x. Because the pATR4 plasmid has a selection marker, hph Žhygromycin B phosphotransferase., after transfection of F9 cells with pATR4, F9 clones, which acquired the plasmid, were selected by their resistance to Hygromycin B and taken for analysis of the recA gene expression. Expression of the modified E. coli recA gene in transgenic F9 cell lines was assayed in Western blots probed with RecA-specific polyclonal rabbit antibodies. T-RecA protein appeared stable in mouse cells and showed the expected molecular weight of about 38 kDa. The levels of T-RecA production varied in the range of 0.05–0.1% of total protein in different clones, as judged by a comparison of staining intensities with those of dilution series of purified RecA protein. The intensity of signal on line 3 ŽFig. 1. is approaching the intensity of those obtained by antibodies detection of about 40 ng of purified RecA protein ŽBoehringer Mannheim.. Thus, the F9–305 clone with the T-RecA production level about 0.1% was chosen for further analysis ŽFig. 1, line 3.. 3.2. Gene targeting is stimulated by nucleus-targeted RecA protein The classical experimental system w5,10,11x was used to measure the frequency of endogenous mouse hprt gene inactivation by homologous recombination with the targeting vector pRV9.1, containing a fragment of hprt gene with insertion of a neo gene. The neo gene disrupts hprt coding sequence in the vector as well as renders cells, which acquired a functional copy of the gene, resistant to the cytotoxic drug
68
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
Fig. 1. Western blot of lysates of the F9 clones resistant to Hygromycin B. Proteins were separated by electrophoresis in 12% polyacrylamide gel containing SDS and RecA-specific protein detected in Western blot with polyclonal anti-RecA antibodies as described in Section 2. Aliquots Ž40 mg of total protein. of the lysates of the F9 clones resistant to Hygromycin B were loaded in lines 2, 3, 4. Lines 1 — E. coli RecA protein ŽBoehringer Mannheim., 1 mg; 3 — F9–305 clone; 2.4 — other Hyg R clones.
G418, by either homologous or random insertion into the host genome. While the hprt gene resides on the X chromosome and homozygotic XY ES cells contain only a single copy of this gene, male mouse embryonic teratocarcinoma cells were used in our experiments. Because the inactivation of hprt gene confers cells resistance to the 6-TG, F9 and F9–305 clones pos-
sessed the resistance to both G418 and 6-TG and were registered as hprt knockout clones. After transfection of F9 as well as F9–305 cells with the pRV9.1 plasmid and selection first for G418 R and then for 6-TG R , we determined the relative frequency of targeted inactivation of hprt gene as the ratio between G418- and 6-TG-resistant clones to the number of G418-resistant clones in five independent experiments. In fact, this is the ratio between homologous and random integration of the plasmid. As shown in Table 1, the frequency of targeted inactivation of hprt gene was, in average, 10-fold higher in the F9–305 cells with overexpression of T-RecA protein then in F9 cells, which are not expressing this recombinase. It is unlikely that the observed increase in frequency of inactivation of hprt gene could be explained by simple variation in the targeting frequencies of different F9 subclones. During our studies Žabout 2 years., we have been estimating the relative frequency of targeted hprt gene inactivation in many F9 subclones. The value of this parameter was not higher than 1:500. To ensure that 6-TG-resistant colonies were not the result of increased level of spontaneous mutation in hprt gene in mouse cells expressing T-RecA, both
Table 1 Relative frequency of targeted inactivation of hprt gene in F9 cells and F9–305 cells with overexpression of T-RecA protein Number of Number of Number of Frequency of Number of Ratio cells surviving cells taken for G418-resistant random integration G418 R q 6-TG R wŽ6-TG R q G418 R .: Ž=10y4 . b electroporation further selection colonies colonies G418 R x 48 h later a no vector 10 7 pRV9.1 1 1 = 10 7 2 3 = 10 7 3 3 = 10 7 4 2 = 10 7 5 1 = 10 7 F9–305 cells no vector 10 7 pRV9.1 1 1.6 = 10 7 2 2 = 10 7 3 3 = 10 7 4 2 = 10 7 5 3 = 10 7 F9 cells
a
5 = 10 6 2.5 = 10 6 3 = 10 6 10 = 10 6 3 = 10 6 1.7 = 10 6 5 = 10 6 2.5 = 10 6 3.5 = 10 6 3 = 10 6 4 = 10 6 6.2 = 10 6
0 1070 2400 8400 2100 1050 0 1750 3500 2860 3200 3100
0
6.77 " 1.55
0
7.9 " 1.49
0 2 4 22 3 2 0 42 67 99 49 49
0 1:535 1:600 1:380 1:700 1:525 0 1:42 1:52 1:30 1:65 1:63
The number of cells which survived electroporation and cultivated 48 h in Eagle medium supplemented with 10% serum. The frequency of random integration of pRV9.1 plasmid calculated as the ratio of the number of G418 R clones to the total number of cells plated for the selection. b
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
cell lines, F9 and F9–305, were subjected to electroporation without pVR9.1 plasmid and selected for 6-TG resistance. No 6-TG-resistant colonies were detected in these controls after plating 5 = 10 6 cells. 3.3. Analysis of the structure of hprt locus in recombinant clones The correct integration of pRV9.1 targeted vector into genomic hprt locus was confirmed by Southern blot analysis of genomic DNA isolated from some Žtwo to three in every experiment. of those doubly resistant to G418 and 6-TG F9 and F9–305 clones using the 32 P-labeled fragments comprising hprt exons 7 Žprobe a. and 9 Žprobe b . ŽFig. 2.. Insertion of the neo gene into exon 8 of host hprt gene must change the structure of hprt locus specifically w11x. Fig. 2a shows restriction map of the wild-type hprt locus, pRV9.1 vector and the same locus after integration of pRV9.1. The 9.1-kb EcoRI fragment containing a wild-type hprt gene from F9 cells was detected by Southern blot using probe a Ž32 P-labeled fragment of exon 7.. The insertion of neo gene into exon 8 will create an additional EcoRI
69
site. This will lead to the hybridization of the probe a with 8.1 kb fragment. As shown on Fig. 2b, a typical Southern blot hybridization of EcoRI-digested DNA from the original F9 cells and F9 cells doubly resistant to G418 and 6-TG with the probe a hprt knockout clones revealed only the predicted 8.1 kb fragment. Southern blot hybridization of XbaI-digested DNA from F9 cells and G418 R , 6-TG R clones with 32 P-labeled fragment of exon 9 revealed the expected alteration of the length of 8.4 kb fragment found in wild-type cells to 9.4 kb found in G418 R , 6-TG R cells ŽFig. 2c.. This could be explained by integration of 1-kb neo gene into exon 8 of hprt gene. Southern blot hybridization of genomic DNA of F9-305 G418 R , 6-TG R clones with probes a and b revealed the same alteration of hprt locus as in F9 G418 R , 6-TG R cells Ždata not shown.. Thus, the inactivation of hprt gene in F9 and F9-395 G418 R , 6-TG R targeted clones was a result of homologous recombination between chromosomal copy of hprt gene and fragment of hprt gene in the targeting vector.
Fig. 2. Gene targeting with pRV9.1 Ža–c.. Ža. Scheme of hprt gene targeting with pRV9.1 targeting vector. The upper restriction map represents wild-type hprt DNA from the parental cell line, F9; the middle map — targeting vector pRV9.1; the lower map depicts the targeted loci from the hprty cell lines. Numbered boxes represent exons; striped box represents the neo gene within exon 8; open boxes represent positions of the probes. E, EcoRI; X, XbaI; B, BglII. Žb. Southern blot of genomic DNA from F9 Žline 1. and F9 G418 R 6-TG R clone Žline 2.. XbaI fragmentsq probe b. Žc. Southern blot of genomic DNA from F9 Žline 1. and F9 G418 R 6-TG R clone Žline 2, 3.. EcoRI fragmentsq probe a.
70
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
4. Discussion In this report, we have demonstrated that the overexpression of T- RecA protein in embryonic mouse teratocarcinoma F9 cells increased the frequency of targeted inactivation of hprt gene. Tenfold stimulation of gene targeting via homologous recombination, observed in our study, looks unexpectedly high because the formation and activation of mammalian ‘‘recombinosome’’ requires interaction of many proteins w18–20x. However, other studies have shown that overexpression of bacterial nucleus-targeted RecA protein markedly increases the level of intrachromosomal somatic recombination and resistance of plant cells to DNA-damaging agent w21x. Taken together, the results of this and our studies suggested that the bacterial RecA protein is able to function efficiently as a recombinase in eukaryotic cell. Also, it has been shown that the overexpression of the proteins involved in homologous recombination in mammalian cells could increase the frequency of homologous recombination events w22–25x. In the recent study, Vispe et al. w22x showed that 2–4-fold overexpression of hamster Rad51 increased the rate of intrachromosomal recombination by a factor of 20 in CHO cells. Moreover, Yanez and Porter w23x observed a 2–3-fold increase in gene targeting and enhanced resistance to ionizing radiation in human cells overexpressing HsRAD51. Overexpression of human RAD52 protein also induces homologous intrachromosomal recombination in cultured monkey cells w24x. Supported by these studies, our data extended this conception by showing that overexpression of bacterial RecA protein, which is functionally similar to Rad51 in mammalian cells, increases the frequency of the homologous targeted events.
Acknowledgements We thank Drs. K. Thomas and M. Capecchi ŽUniversity of Utah, USA. for kindly providing plasmid pRV9.1. We thank Dr. Igor Shevelev and Ekaterina Smirnova for the help with the immunoblot analysis. This research was partially supported by an
International Research Scholar award from the Howard Huges Medical Institute Žgrant 75195546101 to V.A.L... References w1x A. Bradley, P. Hasty, A. Davis, R. Ramirez-Solis, Modifying the mouse: design and desire, Biotechnology ŽNew York. 10 Ž1992. 534–539. w2x P. Hasty, J. Rivera-Petez, C. Chang, A. Bradley, Targeting frequency and integration pattern for insertion and replacement vectors in embryonic stem cells, Mol. Cell Biol. 11 Ž1991. 4509–4517. w3x T. Doetschman, R.G. Gregg, N. Maeda, M.L. Hooper, D.W. Melton, S. Thompson, O. Smithies, Targeted correction of a mutant HPRT gene in mouse embryonic stem cells, Nature 330 Ž1987. 576–578. w4x G. Donoho, M. Jasin, P. Berg, Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells, Mol. Cell Biol. 18 Ž1998. 4070–4078. w5x Ch. Deng, M. Capecchi, Reexamination of gene targeting frequency as a function of extent of homology between the targeting vector and the target locus, Mol. Cell Biol. 12 Ž1992. 3365–3371. w6x S. Kowalczykowski, A. Eggelston, Homologous pairing and DNA strand-exchange proteins, Annu. Rev. Biochem. 63 Ž1994. 991–1043. w7x V. Brendel, L. Brocchieri, S.J. Sandler, A.J. Clark, S. Karlin, Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms, J. Mol. Evol. 44 Ž1997. 528–541. w8x R. Gupta, L. Bazemore, E. Golub, C. Radding, Activities of human recombination protein Rad51, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 463–468. w9x M. Kido, Y. Yoneda, M. Nakanishi, U. Tsuyoshi, Y. Okada, Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells, Exp. Cell Res. 198 Ž1992. 107–114. w10x K. Thomas, M. Capecchi, Site-directed mutagenesis by gen targeting in mouse embryo-derived stem cells, 51 Ž1987. 503–512. w11x K. Thomas, Ch. Deng, M. Capecchi, High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors, Mol. Cell Biol. 12 Ž1992. 2919–2923. w12x T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982. w13x S.A. Bulat, M. Lubeck, N. Mironenko, D.F. Jensen, P.S. Lubeck, UP-PCR analysis and ITS 1 ribotyping of Trichoderma and Gliocladium fungi, Mycol. Res. 102 Ž1998. 933– 943. w14x U. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 Ž1970. 680–685. w15x M. Bradford, A rapid and sensitive method for the quantita-
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
w16x
w17x
w18x
w19x
w20x
tion of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 Ž1976. 248–254. D. Kalderon, B.L. Roberts, W.D. Richardson, A.E. Smith, Sequence requirements for nuclear location of simian virus 40 large T antigen, Nature 311 Ž1984. 33–38. B. Reiss, H. Kosak, M. Klemm, J. Schell, Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells, Mol. Gen. Genet. 253 Ž1997. 695–702. F.E. Benson, P. Baumann, S.C. West, Synergistic actions of Rad51 and Rad52 in recombination and DNA repair, Nature 391 Ž1998. 401–404. E. Golub, O. Kovalenko, R. Gupta, D. Ward, C. Radding, Interaction of human recombination proteins Rad51 and Rad54, Nucleic Acids Res. 25 Ž1997. 4106–4110. E. Golub, R. Gupta, T. Haaf, M. Wold, C. Radding, Interaction of human rad51 recombination protein with singlestranded DNA binding protein, RPA, Nucleic Acids Res. 26 Ž1998. 5388–5393.
71
w21x B. Reiss, M. Klemm, H. Kosak, J. Shell, RecA protein stimulates homologous recombination in plants, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 3094–3098. w22x S. Vispe, C. Cazaux, C. Lesca, M. Defais, Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation, Nucleic Acids Res. 26 Ž1998. 2859–2864. w23x R. Yanez, A. Porter, Gene targeting is enhanced in human cells overexpressing hRAD51, Gene Ther. 6 Ž1999. 1282– 1290. w24x M. Park, Expression of human RAD52 confers resistance to ionizing radiation in mammalian cells, J. Biol. Chem. 270 Ž1995. 15467–15470. w25x B. Johnson, B. Thyagarajan, L. Krueger, B. Hirsch, C. Campbell, Elevated levels of recombinational DNA repair in human somatic cells expressing the Saccharomyces cereÕisiae RAD52 gene, Mutat. Res. 363 Ž1996. 179–189.
Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells Olga G. Shcherbakova a , Vladislav A. Lanzov a , Hideyuki Ogawa b, Michael V. Filatov a,) a
Molecular and Radiation Biophysics DiÕision, Petersburg Nuclear Physics Institute, Gatchina 188350, Russian Federation b Department of Biology, Osaka UniÕersity, Osaka, Japan Received 18 May 1999; received in revised form 14 October 1999; accepted 22 October 1999
Abstract The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells. q 2000 Elsevier Science B.V. All rights reserved. Keywords: RecA protein; Gene targeting; Homologous recombination in somatic mammalian cells
1. Introduction Gene targeting in mouse embryonic stem ŽES. cells, if successful, provides a powerful method for systemic alteration of mammalian genome as well as for studying gene functions in vivo w1x. Different experimental strategies for targeted gene modification in the desired chromosomal loci of ES cells via
) Corresponding author. Tel.: q7-81271-46626; fax: q781271-32303; e-mail: [email protected]
homologous recombination have been developed recently. The major difficulty in general application of these strategies is the fact that homologous recombination in ES cells is an infrequent event w2,3x. Several factors — the length of homologous fragment in input DNA w5x, the presence of double strand breaks in the targeted genomic locus w4x, the usage of targeting vectors bearing DNA isogenic to DNA of the host cells w5x — are influenced much on frequency of homologous targeted events. The introduction of an active exogenous recombinase is one of the possible approaches to increase the
0921-8777r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 5 9 - 2
66
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
efficiency of the gene targeting in mammalian cells. The Escherichia coli RecA protein has been shown to be responsible for two main steps of recombination process: the search for homology and for strand exchange between two DNA molecules w6x. A number of proteins with structural and functional similarity to RecA have been found in three major domains of life — Prokarya, Archaea and Eukarya w7x. A human HsRad51 protein has been shown to possess both DNA-dependent ATPase activity and capability to form a nucleoprotein filament that resembles that of RecA w8x. Although the rates of ATP hydrolysis, homologous pairing and subsequent strand exchange promoted by the HsRad51 appear to be less efficient, the functional and structural similarity of HsRad51 and RecA becomes obvious. These facts prompted us to study the possibility of whether the additional production of bacterial RecA protein in mammalian cells can specifically increase the frequency of homologous recombination. For this aim, the E. coli RecA protein fused with a nuclear localization signal ŽNLS. was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of targeted inactivation of hprt gene was 10-fold increased in the mouse cells expressing the RecA protein.
2. Materials and methods 2.1. Plasmids The pATR4 plasmid was a kind gift of Dr. K. Kohno ŽInstitute for Molecular and Cellular Biology, Osaka, Japan.. The plasmid contains the recA gene fused with the NLS of the SV40 large T antigen ŽPro–Lys–Lys–Lys–Arg–Lys–Val–Glu–Asp–Pro. that specifies the nuclear translocation of the SV40 large T antigen w16x. The expression of the recA gene in mammalian cells was provided by replacement of original recA promoter with chicken b-actin promoter and Kozak’s sequence in pATR4 plasmid w9x. This plasmid also bears the selection marker, hph Žhygromycin B phosphotransferase. w9x. The pVR9.1 plasmid, kindly provided by Drs. K. Thomas and M. Capecchi ŽUniversity of Utah, USA.,
contains the 9.1-kb fragment of hprt gene with insertion of the selectable marker, neo gene w5,10,11x. In order to generate targeting vector, the pVR9.1 plasmid was cleaved by BglII restriction endonuclease. 2.2. Cell culture, transfection, and selection of drug-resistant cells Mouse embryonic teratocarcinoma cells, line F9, were grown in L-glutamine containing Eagle medium ŽBiolot, Russia. supplemented with 10% bovine serum ŽBiolot, Russia. and gentamycin sulphate ŽSigma.. DNA was introduced into the F9 cells by electroporation. Prior electroporation growing cells were trypsinised, washed with Eagle medium and counted. In each experiment, 1.8 mg of DNA ŽpRV9.1, linearized by BglII. has been used per 1 = 10 6 cells taken for electroporation. Cells were mixed with DNA and electroporated at 1.08 kV and 0.2 mF. Under these conditions, 50 " 10% of the cells survived the procedure. Then cells were placed in prewarmed complete culture medium and 48 h later cells were trypsinised and counted. The 2–4 = 10 5 cells per flask were plated in the complete medium supplemented with 500 mgrml G418 ŽSigma.. The cells were maintained under G418 selection for 9–11 days, until G418 R colonies have become visible. The colonies in control flasks were fixed and counted in each experiment. After selection with G418, the growth medium was supplemented with 0.2 mgrml 6-thioguanine Ž6-TG; Sigma. and cells were maintained about 10 days of the selection. At the end of this period, the number of colonies, doubly resistant to G418 and 6-TG, was determined. 2.3. Southern blot analysis F9 cell lines resistant to G418 and 6-TG were grown under the standard condition. Genomic DNA was purified from the cells using a standard procedure w12x. These DNAs were digested with EcoRI or XbaI enzyme and separated on 0.8% agarose gel. DNA was transferred to Hybond-N ŽAmersham. nylon membrane by alkali transfer protocol w12x, prehybridised with 6 = SSPE, 0.2% SDS, 10 mgrml
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
heparin ŽSpofa., and hybridised in the same buffer for 17 h at 658C with probes a or b w13x. Probe a is a 32 P-labeled single-stranded fragment of DNA, obtained by one-primer polymerase chain reaction ŽPCR. amplification with the primer. 5X-TCGAGAGGTCCTTTTCACC used 800 bp EcoRI–BstEII fragment of pRV9.1 vector containing part of exon 7 of hprt gene as the template. Probe b was a 32 P-labeled single-stranded fragment of DNA obtained in PCR reaction with the primer, 5X-TCCTGTTGGATGTTGCCAGTAAA using the 1000bp EcoRI fragment of pRV9.1 vector containing part of exon 9 of hprt gene as the template. Probes a and b were used for hybridization with EcoRI- and XbaI-digested genomic DNAs correspondingly. 32 P signals were revealed by autoradiography with the enhancing screen at y708C. 2.4. Production of the cell lines expressing T-RecA protein DNA of pATR4 plasmid was electroporated into F9 cells by the procedure described above. Fortyeight hours after electroporation, cells were plated in Eagle medium, supplemented with 10% bovine serum and 400 mgrml Hygromycin B ŽSigma.. After 10 days of the selection, colonies resistant to the Hygromycin B, and therefore harboring pATR4 plasmid in their genomes, were picked up and grown under the standard conditions. When cells reached confluence, they were trypsinised, harvested and resuspended in Laemmli w14x sample buffer without bromophenol blue. After heat denaturation, lysates were clarified by centrifugation and protein concentration was determined by Bradford w15x method. About 20–40 mg of protein per line was subjected to 12.5% polyacrylamide SDS gels w14x. Resolved proteins were transferred to nitrocellulose membranes by the standard protocol w12x. RecA protein was revealed using the polyclonal rabbit anti-RecA antibodies in concentration 0.1 mgrml in PBS-0.05% Tween-20 buffer with 5% non-fat dry milk as blocking reagent. The first antibody against RecA protein was raised in rabbits by repeated intradermal injections of purified RecA protein with Freind’s adjuvant ŽCalbiochem, Behring, La Jolla.. Antigen–antibodies complexes were visualized by peroxidase-conjugated swine anti rabbit immunoglobulins ŽDako..
67
3. Results 3.1. Generation of mouse cell line expressing nucleus-targeted RecA protein In order to investigate the influence of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, pATR4 plasmid containing the E. coli RecA protein encoding gene fused to NLS from large T antigen of SV40, described above, was introduced into the mouse F9 teratocarcinoma cells. The RecA protein modified by fusion with the translocation signal ŽT-RecA. has been shown to keep the original activity of the RecA protein and to be efficiently transported into the nuclei of mammalian cells w9,17x. Because the pATR4 plasmid has a selection marker, hph Žhygromycin B phosphotransferase., after transfection of F9 cells with pATR4, F9 clones, which acquired the plasmid, were selected by their resistance to Hygromycin B and taken for analysis of the recA gene expression. Expression of the modified E. coli recA gene in transgenic F9 cell lines was assayed in Western blots probed with RecA-specific polyclonal rabbit antibodies. T-RecA protein appeared stable in mouse cells and showed the expected molecular weight of about 38 kDa. The levels of T-RecA production varied in the range of 0.05–0.1% of total protein in different clones, as judged by a comparison of staining intensities with those of dilution series of purified RecA protein. The intensity of signal on line 3 ŽFig. 1. is approaching the intensity of those obtained by antibodies detection of about 40 ng of purified RecA protein ŽBoehringer Mannheim.. Thus, the F9–305 clone with the T-RecA production level about 0.1% was chosen for further analysis ŽFig. 1, line 3.. 3.2. Gene targeting is stimulated by nucleus-targeted RecA protein The classical experimental system w5,10,11x was used to measure the frequency of endogenous mouse hprt gene inactivation by homologous recombination with the targeting vector pRV9.1, containing a fragment of hprt gene with insertion of a neo gene. The neo gene disrupts hprt coding sequence in the vector as well as renders cells, which acquired a functional copy of the gene, resistant to the cytotoxic drug
68
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
Fig. 1. Western blot of lysates of the F9 clones resistant to Hygromycin B. Proteins were separated by electrophoresis in 12% polyacrylamide gel containing SDS and RecA-specific protein detected in Western blot with polyclonal anti-RecA antibodies as described in Section 2. Aliquots Ž40 mg of total protein. of the lysates of the F9 clones resistant to Hygromycin B were loaded in lines 2, 3, 4. Lines 1 — E. coli RecA protein ŽBoehringer Mannheim., 1 mg; 3 — F9–305 clone; 2.4 — other Hyg R clones.
G418, by either homologous or random insertion into the host genome. While the hprt gene resides on the X chromosome and homozygotic XY ES cells contain only a single copy of this gene, male mouse embryonic teratocarcinoma cells were used in our experiments. Because the inactivation of hprt gene confers cells resistance to the 6-TG, F9 and F9–305 clones pos-
sessed the resistance to both G418 and 6-TG and were registered as hprt knockout clones. After transfection of F9 as well as F9–305 cells with the pRV9.1 plasmid and selection first for G418 R and then for 6-TG R , we determined the relative frequency of targeted inactivation of hprt gene as the ratio between G418- and 6-TG-resistant clones to the number of G418-resistant clones in five independent experiments. In fact, this is the ratio between homologous and random integration of the plasmid. As shown in Table 1, the frequency of targeted inactivation of hprt gene was, in average, 10-fold higher in the F9–305 cells with overexpression of T-RecA protein then in F9 cells, which are not expressing this recombinase. It is unlikely that the observed increase in frequency of inactivation of hprt gene could be explained by simple variation in the targeting frequencies of different F9 subclones. During our studies Žabout 2 years., we have been estimating the relative frequency of targeted hprt gene inactivation in many F9 subclones. The value of this parameter was not higher than 1:500. To ensure that 6-TG-resistant colonies were not the result of increased level of spontaneous mutation in hprt gene in mouse cells expressing T-RecA, both
Table 1 Relative frequency of targeted inactivation of hprt gene in F9 cells and F9–305 cells with overexpression of T-RecA protein Number of Number of Number of Frequency of Number of Ratio cells surviving cells taken for G418-resistant random integration G418 R q 6-TG R wŽ6-TG R q G418 R .: Ž=10y4 . b electroporation further selection colonies colonies G418 R x 48 h later a no vector 10 7 pRV9.1 1 1 = 10 7 2 3 = 10 7 3 3 = 10 7 4 2 = 10 7 5 1 = 10 7 F9–305 cells no vector 10 7 pRV9.1 1 1.6 = 10 7 2 2 = 10 7 3 3 = 10 7 4 2 = 10 7 5 3 = 10 7 F9 cells
a
5 = 10 6 2.5 = 10 6 3 = 10 6 10 = 10 6 3 = 10 6 1.7 = 10 6 5 = 10 6 2.5 = 10 6 3.5 = 10 6 3 = 10 6 4 = 10 6 6.2 = 10 6
0 1070 2400 8400 2100 1050 0 1750 3500 2860 3200 3100
0
6.77 " 1.55
0
7.9 " 1.49
0 2 4 22 3 2 0 42 67 99 49 49
0 1:535 1:600 1:380 1:700 1:525 0 1:42 1:52 1:30 1:65 1:63
The number of cells which survived electroporation and cultivated 48 h in Eagle medium supplemented with 10% serum. The frequency of random integration of pRV9.1 plasmid calculated as the ratio of the number of G418 R clones to the total number of cells plated for the selection. b
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
cell lines, F9 and F9–305, were subjected to electroporation without pVR9.1 plasmid and selected for 6-TG resistance. No 6-TG-resistant colonies were detected in these controls after plating 5 = 10 6 cells. 3.3. Analysis of the structure of hprt locus in recombinant clones The correct integration of pRV9.1 targeted vector into genomic hprt locus was confirmed by Southern blot analysis of genomic DNA isolated from some Žtwo to three in every experiment. of those doubly resistant to G418 and 6-TG F9 and F9–305 clones using the 32 P-labeled fragments comprising hprt exons 7 Žprobe a. and 9 Žprobe b . ŽFig. 2.. Insertion of the neo gene into exon 8 of host hprt gene must change the structure of hprt locus specifically w11x. Fig. 2a shows restriction map of the wild-type hprt locus, pRV9.1 vector and the same locus after integration of pRV9.1. The 9.1-kb EcoRI fragment containing a wild-type hprt gene from F9 cells was detected by Southern blot using probe a Ž32 P-labeled fragment of exon 7.. The insertion of neo gene into exon 8 will create an additional EcoRI
69
site. This will lead to the hybridization of the probe a with 8.1 kb fragment. As shown on Fig. 2b, a typical Southern blot hybridization of EcoRI-digested DNA from the original F9 cells and F9 cells doubly resistant to G418 and 6-TG with the probe a hprt knockout clones revealed only the predicted 8.1 kb fragment. Southern blot hybridization of XbaI-digested DNA from F9 cells and G418 R , 6-TG R clones with 32 P-labeled fragment of exon 9 revealed the expected alteration of the length of 8.4 kb fragment found in wild-type cells to 9.4 kb found in G418 R , 6-TG R cells ŽFig. 2c.. This could be explained by integration of 1-kb neo gene into exon 8 of hprt gene. Southern blot hybridization of genomic DNA of F9-305 G418 R , 6-TG R clones with probes a and b revealed the same alteration of hprt locus as in F9 G418 R , 6-TG R cells Ždata not shown.. Thus, the inactivation of hprt gene in F9 and F9-395 G418 R , 6-TG R targeted clones was a result of homologous recombination between chromosomal copy of hprt gene and fragment of hprt gene in the targeting vector.
Fig. 2. Gene targeting with pRV9.1 Ža–c.. Ža. Scheme of hprt gene targeting with pRV9.1 targeting vector. The upper restriction map represents wild-type hprt DNA from the parental cell line, F9; the middle map — targeting vector pRV9.1; the lower map depicts the targeted loci from the hprty cell lines. Numbered boxes represent exons; striped box represents the neo gene within exon 8; open boxes represent positions of the probes. E, EcoRI; X, XbaI; B, BglII. Žb. Southern blot of genomic DNA from F9 Žline 1. and F9 G418 R 6-TG R clone Žline 2.. XbaI fragmentsq probe b. Žc. Southern blot of genomic DNA from F9 Žline 1. and F9 G418 R 6-TG R clone Žline 2, 3.. EcoRI fragmentsq probe a.
70
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
4. Discussion In this report, we have demonstrated that the overexpression of T- RecA protein in embryonic mouse teratocarcinoma F9 cells increased the frequency of targeted inactivation of hprt gene. Tenfold stimulation of gene targeting via homologous recombination, observed in our study, looks unexpectedly high because the formation and activation of mammalian ‘‘recombinosome’’ requires interaction of many proteins w18–20x. However, other studies have shown that overexpression of bacterial nucleus-targeted RecA protein markedly increases the level of intrachromosomal somatic recombination and resistance of plant cells to DNA-damaging agent w21x. Taken together, the results of this and our studies suggested that the bacterial RecA protein is able to function efficiently as a recombinase in eukaryotic cell. Also, it has been shown that the overexpression of the proteins involved in homologous recombination in mammalian cells could increase the frequency of homologous recombination events w22–25x. In the recent study, Vispe et al. w22x showed that 2–4-fold overexpression of hamster Rad51 increased the rate of intrachromosomal recombination by a factor of 20 in CHO cells. Moreover, Yanez and Porter w23x observed a 2–3-fold increase in gene targeting and enhanced resistance to ionizing radiation in human cells overexpressing HsRAD51. Overexpression of human RAD52 protein also induces homologous intrachromosomal recombination in cultured monkey cells w24x. Supported by these studies, our data extended this conception by showing that overexpression of bacterial RecA protein, which is functionally similar to Rad51 in mammalian cells, increases the frequency of the homologous targeted events.
Acknowledgements We thank Drs. K. Thomas and M. Capecchi ŽUniversity of Utah, USA. for kindly providing plasmid pRV9.1. We thank Dr. Igor Shevelev and Ekaterina Smirnova for the help with the immunoblot analysis. This research was partially supported by an
International Research Scholar award from the Howard Huges Medical Institute Žgrant 75195546101 to V.A.L... References w1x A. Bradley, P. Hasty, A. Davis, R. Ramirez-Solis, Modifying the mouse: design and desire, Biotechnology ŽNew York. 10 Ž1992. 534–539. w2x P. Hasty, J. Rivera-Petez, C. Chang, A. Bradley, Targeting frequency and integration pattern for insertion and replacement vectors in embryonic stem cells, Mol. Cell Biol. 11 Ž1991. 4509–4517. w3x T. Doetschman, R.G. Gregg, N. Maeda, M.L. Hooper, D.W. Melton, S. Thompson, O. Smithies, Targeted correction of a mutant HPRT gene in mouse embryonic stem cells, Nature 330 Ž1987. 576–578. w4x G. Donoho, M. Jasin, P. Berg, Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells, Mol. Cell Biol. 18 Ž1998. 4070–4078. w5x Ch. Deng, M. Capecchi, Reexamination of gene targeting frequency as a function of extent of homology between the targeting vector and the target locus, Mol. Cell Biol. 12 Ž1992. 3365–3371. w6x S. Kowalczykowski, A. Eggelston, Homologous pairing and DNA strand-exchange proteins, Annu. Rev. Biochem. 63 Ž1994. 991–1043. w7x V. Brendel, L. Brocchieri, S.J. Sandler, A.J. Clark, S. Karlin, Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms, J. Mol. Evol. 44 Ž1997. 528–541. w8x R. Gupta, L. Bazemore, E. Golub, C. Radding, Activities of human recombination protein Rad51, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 463–468. w9x M. Kido, Y. Yoneda, M. Nakanishi, U. Tsuyoshi, Y. Okada, Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells, Exp. Cell Res. 198 Ž1992. 107–114. w10x K. Thomas, M. Capecchi, Site-directed mutagenesis by gen targeting in mouse embryo-derived stem cells, 51 Ž1987. 503–512. w11x K. Thomas, Ch. Deng, M. Capecchi, High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors, Mol. Cell Biol. 12 Ž1992. 2919–2923. w12x T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982. w13x S.A. Bulat, M. Lubeck, N. Mironenko, D.F. Jensen, P.S. Lubeck, UP-PCR analysis and ITS 1 ribotyping of Trichoderma and Gliocladium fungi, Mycol. Res. 102 Ž1998. 933– 943. w14x U. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 Ž1970. 680–685. w15x M. Bradford, A rapid and sensitive method for the quantita-
O.G. ShcherbakoÕa et al.r Mutation Research 459 (2000) 65–71
w16x
w17x
w18x
w19x
w20x
tion of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 Ž1976. 248–254. D. Kalderon, B.L. Roberts, W.D. Richardson, A.E. Smith, Sequence requirements for nuclear location of simian virus 40 large T antigen, Nature 311 Ž1984. 33–38. B. Reiss, H. Kosak, M. Klemm, J. Schell, Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells, Mol. Gen. Genet. 253 Ž1997. 695–702. F.E. Benson, P. Baumann, S.C. West, Synergistic actions of Rad51 and Rad52 in recombination and DNA repair, Nature 391 Ž1998. 401–404. E. Golub, O. Kovalenko, R. Gupta, D. Ward, C. Radding, Interaction of human recombination proteins Rad51 and Rad54, Nucleic Acids Res. 25 Ž1997. 4106–4110. E. Golub, R. Gupta, T. Haaf, M. Wold, C. Radding, Interaction of human rad51 recombination protein with singlestranded DNA binding protein, RPA, Nucleic Acids Res. 26 Ž1998. 5388–5393.
71
w21x B. Reiss, M. Klemm, H. Kosak, J. Shell, RecA protein stimulates homologous recombination in plants, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 3094–3098. w22x S. Vispe, C. Cazaux, C. Lesca, M. Defais, Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation, Nucleic Acids Res. 26 Ž1998. 2859–2864. w23x R. Yanez, A. Porter, Gene targeting is enhanced in human cells overexpressing hRAD51, Gene Ther. 6 Ž1999. 1282– 1290. w24x M. Park, Expression of human RAD52 confers resistance to ionizing radiation in mammalian cells, J. Biol. Chem. 270 Ž1995. 15467–15470. w25x B. Johnson, B. Thyagarajan, L. Krueger, B. Hirsch, C. Campbell, Elevated levels of recombinational DNA repair in human somatic cells expressing the Saccharomyces cereÕisiae RAD52 gene, Mutat. Res. 363 Ž1996. 179–189.