Nucleic Acids Research Advance Access originally published online on November 10, 2006
Nucleic Acids Research 2006 34(21):6298-6304; doi:10.1093/nar/gkl890
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Nucleic Acids Research, 2006, Vol. 34, No. 21 6298-6304
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
DAXX interacts with phage 
C31 integrase and inhibits recombination
1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University China 2 Department of Surgery, University of British Columbia Vancouver, Canada
*To whom correspondence should be addressed. Tel/Fax: +86 21 65649899; Email: Jlxue{at}fudan.ac.cn
Received July 10, 2006. Revised October 8, 2006. Accepted October 9, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phage
C31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between C31 integrase and cellular proteins have never been investigated. Using pLexA-C31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 106 independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and C31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for C31 binding. Hybridization between a C31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of C31 is responsible for the interaction with DAXX. This tetramer is also necessary for C31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with C31 integrase in a HEK293-derived C31 integrase activity reporter cell line significantly reduced the C31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with C31 causing a mild inhibition in the integration efficiency. This is the first time that C31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene therapy remains the most promising if not the only approach to treating genetic diseases. The safety concerns of gene therapy have been heightened by the discovery of a leukemia-like disorder in several patients treated with a retrovirus vector, due to an insertional mutagenesis (1). To minimize the potential risk of gene therapy, therapeutic genes should be integrated at specific sites proven to be safe.
While homologous recombination gives rise to precise site-specificity, the low efficiency limits its clinical application (2). Alternatively, integrases mediate site-specific insertions into the genome by recognizing specific sequences. Site-specific recombination systems are ubiquitous throughout prokaryotes but are rare in mammals (3). Some phage recombinases such as Cre are able to insert a gene into a specific site (loxP) and have been employed in engineering mammalian cells (4,5). However, these recombination systems require pre-insertion of their substrate DNA sequences, such as the loxP site for Cre, located in the target region in mammalian genomes, which limits their potential clinical applications. Phage C31 integrase belongs to the serine recombinase family that mediates site-specific recombination between two short recognition sites, attB and attP (3,6). Recombination occurs when these two att sites are present in two different DNA molecules and the attB serves as the donor to be inserted into the attP (3). Interestingly, it has been discovered that C31 integrase can also mediate recombination at a limited number of sites in mammalian genomes (7). These ‘pseudo-att’ sites have been proposed as potential targets for site-specific gene insertion using the C31 integrase-based system (8–11). Several studies have demonstrated the usefulness of the C31 integrase system in gene therapy. A C31-integrase-mediated gene insertion into a human myoblast genome resulted in a 15-fold increase in stable expression compared to that without integrase (12). Similarly, coinjection of plasmid-expressing C31 in the mdx mouse resulted in 5- to 10-fold higher levels of sustained luciferase expression as well as dystrophin expression (13). However, potential interactions between C31 and proteins in mammalian host cells have never been studied carefully. This issue has become particularly important as it has been reported that C31 integrase induced a high frequency of hepatocyte dysplasia after the integrase system was used to transfer the fumarylacetoacetate hydrolase (FAH) gene into the livers of mice affected with hereditary tyrosinemia type 1 (14).
To test the hypothesis that C31 integrase may interact with cellular proteins, we employed a yeast-two-hybridization assay to detect cellular proteins that interact with C31 integrase. We report here that C31 can strongly bind to an important cellular protein, DAXX, as evidenced by the results of yeast-two-hybridization and co-immunoprecipitation. We have identified the binding regions in both proteins and the functional interaction between DAXX and C31 were also demonstrated by reduced C31 integrase activity in DAXX-overexpressing cells.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid construction
To construct a pLexA-
C31 bait plasmid, the open reading frame (ORF) of C31 integrase was amplified from a pCMVInt plasmid (kindly provided by Prof. M. P. Calos) with a pair of primers: 5'-gaggatcctgacacaaggggttgtgac-3' and 5'-ccgctcgagcgccgctacgtcttccgtg-3'. The PCR product was inserted into the plasmid pLexA (Clontech) between the BamHI and XhoI sites. The N-terminal catalytic-activity-containing fragment (1–235 amino acid toward CAC93948
[GenBank]
) and C-terminal fragment (237–613 amino acid towards CAC93948
[GenBank]
) were amplified with primers 5'-gaggatcctgacacaaggggttgtgac-3', 5'-ccgctcgagcgcttacaaagccccgtgatgctg-3' and 5'-gaggatcctggacgctgacgccgtgccg-3', 5'-ccgctcgagcgccgctacgtcttccgtg-3'. The fragments were then inserted into the pLexA plasmid between BamHI and XhoI to produce the plasmids pLexA-C31 (1–235) and pLexA-C31 (237–613). Both these plasmids were used in a yeast-mating test for locating the interacting fragment of C31 integrase.
pB42AD-DAXX(572–625) and pB42AD-DAXX(625–740) were used for determining the C31-interacting fragment on DAXX. These fragments were amplified from a pB42AD fetal brain cDNA library (Clontech) with the primers 5'-gcgaattcaacatggaagctttgcccctggatac-3', 5'-ccgctcgagcggaccagaatctccccagttgtg-3' and 5'-gcgaattcaacatgtctggtcccccctgcaaaaaatc-3', 5'-ggagacttgaccaaacctctggcg-3'. The PCR products were inserted into the site between EcoRI and XhoI in the pB42AD plasmid (Clontech).
The above obtained C31 ORF was also subcloned into pEGFP-C2 (Clontech) between BamHI and XhoI sites in the frame with the C-terminus of EGFP to generate pEGFP-C31 that expresses EGFP-C31 fusion protein. A construct pC31-EGFP expressing C-terminal fused C31 fusion protein was produced similarly.
The ORF of human DAXX was obtained from a human fetal brain cDNA library (Clontech) by amplification with primers 5'-gagaattcacaatggccaccgctaacagc-3' and 5'-gaggatcctcagagtctgagagcacg-3'. The cDNA was inserted into pDsRed-N1 (Clontech) between EcoRI and BamHI to generate pDAXX-DsRed that expresses DAXX-DsRed fusion protein. Plasmids that express mutant C31s, namely, pLexA-C31(Del451–454), pLexA-C31(R451H) and pLexA-C31 (K454N), were generated by a PCR-based point specific mutation strategy with the primers shown in Table 1.
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The above mutant
C31s were also amplified with primers 5'-gaggatcctatgacacaaggggttgtgac-3' and 5'-ccgctcgagcgccgctacgtcttccgtg-3' and inserted into pcDNA3.0 (Invitrogen) between BamHI and XhoI for expression and determination of the catalytic activity of mutant C31. The plasmids mentioned above were all verified by sequencing analysis.
Yeast-two-hybridization assay
The MATCHMARKER LexA two-hybrid system was purchased from Clontech. Yeast-two-hybridization was performed in a standard two-step protocol provided by the manufacturer. All isolated positive clones were amplified with the uniform pB42AD sequence primers (5'-ccagcctcttgctgagtggagatg-3'; 5'-ggagacttgaccaaacctctggcg-3') and the PCR products were sequenced with the same primers.
For locating the interacting fragment on DAXX, yeast-mating tests between pB42AD-DAXX(572–625)/pB42AD-DAXX(627–740)-transferred EGY48[p8opLacZ] and pLexA-C31-transferred YM4271 were performed according to a standard yeast-mating protocol provided by the manufacturer.
The yeast-mating test was also used to determine the interacting fragment on C31 integrase. Either pLexA-C31(1–235), pLexA-C31(237–613), pLexA-C31(Del451–454), pLexA-C31(R451H) or pLexA-C31(K454N) was used to correspondingly transfect YM4271, which was then mated with pB42AD-DAXX(527–740) EGY48[p8opLacZ] yeast cells.
Co-immunoprecipitation
Co-immunoprecipitation was employed to verify the interaction between C31 integrase and cellular protein DAXX. The HEK293 cells were maintained in DMEM (Gibco) medium with 10% fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. The cells were transfected with pEGFP-C31 or a control plasmid pEGFP-N3 (Clontech) using Lipofectamine (Invitrogen). At 24 h post-transfection, the cells were harvested and lysed with 1 ml lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 0.5 mM PMSF, 0.5% protease inhibitor cocktail (Sigma)). Ten micrograms of mouse anti-GFP monoclonal antibody (NeoMarkers) were added to 1 ml of each cell lysate. After incubation with end-over-end mixing for 2 h at 4°C, 10 µl of pre-washed protein A/G Sepharose (Santa Cruz) were added to each extract and shaken for 1 h at 4°C. Next, the immunoprecipitates were washed three times with the lysis buffer. The pellets were then separated on SDS–PAGE and transferred to a nitrocellulose membrane. The blot was detected using rabbit anti-human DAXX antibody (Santa Cruz) followed by horseradish-peroxidase-conjugated anti-rabbit antibody (Sigma) and the bands were visualized using an enhanced chemiluminescence immunoblotting detection kit (Amersham).
C31 peptide array analysis
Three C31 peptide arrays (12, 15 and 20 mers) were generated and probed with DAXX to determine the DAXX binding region in C31. Cellulose-bound overlapping peptides derived from C31 integrase were synthesized using an AutoSpot robot (Intavis Bioanalytical Instruments).
The basic lengths of the spotted fragments for the three arrays were 12, 15 and 20 residues, respectively. Each neighboring peptide shifted two residues towards the C-terminus. The arrays were incubated with a cell lysate of 5 x 107 HEK293 cells in 10 ml of lysis buffer at 4°C overnight. The membranes were then incubated with a rabbit anti-human DAXX polyclonal antibody (Santa Cruz) followed by horseradish-peroxidase-conjugated anti-rabbit antibody (Sigma). The bound proteins were visualized using enhanced chemiluminescence immunoblotting detection reagents (Amersham). For controls, the membranes were processed identically but incubation with the cell lysate was omitted.
Intracellular localization of DAXX and C31 integrase
The subcellular locations of DAXX and C31 integrase were determined in HEK293 cells. Fusion plasmids, pDAXX-DsRed, pEGFP-C31 and pC31-EGFP were used to transfer HEK293 for the location of C31 integrase and DAXX, respectively. The cells were transfected with the plasmids above using Lipofectamine (Invitrogen). At 24 h post-transfection, the cells were observed under a confocal fluorescent microscope (TSC SP2, Leica). The nuclei were strained with DAPI as a control. The fluorescence of GFP, RFP and DAPI was observed at wavelengths of 488, 543 and 405 for excitation and 510, 582 and 461 for emission, respectively.
C31 integrase activity assay
C31 integrase activity in Escherichia coli was determined using a pBCPB-(kindly provided by Prof. M.P. Calos) based system (8). Briefly, plasmid containing C31 integrase or its mutant cDNAs was co-transformed with pBCPB into E.coli DH5
-competent cells. The cells were plated on an LB agar medium with ampicillin (50 µg/ml), chloramphenicol (50 µg/ml) and X-gal (40 µg/ml). Recombination through active C31 integrase causes deletion of the beta-glactosidase gene from pBCPB and gives rise to white colonies. The ratio of the number of white colonies to that of total colonies was calculated 24 h later as an index of integrase activity.
C31-mediated integration in mammalian cells was measured using an HEK293-derived C31 integration reporter cell line, PB[EGFP], which was created in our laboratory. PB[EGFP] contains a genomically inserted single copy structure of pCMV-attB-BGH poly A-attP-EGFP. In the presence of C31, integrase-mediated recombination between attB and attP sites removes bGH poly A from the inserted structure, resulting in EGFP expression. At 48 h post-transfection, the cells were harvested and percentages of EGFP-expressing cells were determined by flow cytometery. Overexpression of DAXX was accomplished by co-transfecting pDAXX-DsRed1 with pCMVInt. The plasmid pDsRed1-N1 that contains no DAXX was used as a control construct. Meanwhile, Endogenous expression of DAXX was knocked down by transfection with a DAXX-specific duplex RNA (5'-GGAGUUGGAUCUCUCAGAAdTdT-3'; 5'-UUCUGAGAGAUCCAACUCCdTdT-3') (15), which reduced the DAXX protein expression level by 
75%, as estimated by western blot analysis with a human DAXX-specific antibody. As a control, a human non-silencing duplex RNA (5'-UUAAGUAGCUUGGCCUUGAdTdT-3'; 5'-UCAAGGCCAAGCUACUUAAdTdT-3')(GeneChem, China) was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Results of yeast-two-hybridization and co-immunoprecipitation indicated a strong interaction between
C31 and DAXXWe initially used pLexA-
C31 as a bait to screen a pB42AD-based human fetal brain cDNA library for potential C31 integrase interacting proteins. Sixty-one positives were isolated from 106 independently screened clones and sequenced. Fifty-one out of the 61 positive clones corresponded with the C-terminus of DAXX sequence, from 527 to 730 amino acid (Figure 1A).
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The interaction between
C31 integrase and DAXX was verified with co-immunoprecipitation analysis. HEK293 cells were transfected with either pEGFP-C31 that expressed an EGFP-C31 fusion protein or a control construct pEGFP-N3 containing the EGFP only. A mouse anti-GFP antibody was able to precipitate DAXX from the cell lysate of HEK293 transfected with pEGFP-C31 but not with pEGFP-N3 (Figure 1B).
Characterization of interacting fragments of C31 integrase and DAXX
We then attempted to locate the key fragment that mediates the interaction between C31 integrase and DAXX. Both the C-terminus (237–613) and N-terminus (1–235) of C31 protein were fused to the LexA DNA binding domain. The yeast-mating test revealed that the C- but not the N-terminus of the integrase is necessary for the interaction. To further narrow down the exact region in the C-terminus of C31, peptide arrays displaying overlapping fragments of C31 that covered the entire sequence of the C31 protein were utilized for the binding of endogenous DAXX from the HEK293 cell lysate. Following incubation with the lysate, the bound DAXX was detected with an anti-DAXX antibody. The strongest immunoreactivity was found predominantly at spot 222–226 on 12-residue arrays, spot 221–226 on 15-residue arrays and spot 218–226 on 20-residue arrays (Figure 1C). Interestingly, the positive spots from the three C31 arrays shared a common corresponding region of a tetramer, 451RFGK454, indicating that it was probably the fragment in C31 responsible for DAXX binding (Figure 1D).
For further characterizing the binding site of C31, we generated three mutant C31 constructs, including an Arg451–His451 point mutation (pLexA-C31(R451H)), a Lys454–Asn454 point mutation (pLexA-C31(K454N)) and one with a deletion of the tetramer 451–454 (pLexA-C31(Del451–454)). The yeast-two-hybrid assay showed no interaction between pLexA-C31(Del451–454) and DAXX. However, mutations at the Arg451 (pLexA-C31(R451H)) or Lys454 (pLexA-C31(K454N)) residue of C31 did not alter its binding to DAXX. (Figure 1A).
To localize the region in DAXX that binds to C31, a pB42AD construct containing a DAXX with a deletion of amino acid 627–740 was generated for the yeast-mating test. Deletion of the 627–740 amino acid fragment in DAXX completely abolished its binding to C31 (Figure 1A). Furthermore, a construct containing a fragment of DAXX (627–740) (pB42AD-DAXX (627–740), Figure 1A) was sufficient for binding to C31. These results clearly demonstrated that the tetramer 451RFGK454 in the C31 integrase and the region between 627 and 740 amino acid in DAXX are crucial for the interaction between the two proteins.
Intracellular localization of C31 and DAXX
Plasmids of pDAXX-DsRed, pEGFP-C31, pC31-EGFP and pEGFP that express DAXX-DsRed, N- or C-terminal fused EGFP/C31 fusion proteins and EGFP alone, respectively, were used for intracellular localization of C31 integrase and DAXX. As shown in Figure 2A, punctates of red fluorescent DAXX-DsRed fusion protein were present predominantly in the nucleus. In contrast, both N- and C-terminal fused EGFP/C31 fusion protein were mainly localized in the cytoplasm (Figure 2A–C). Meanwhile, EGFP alone was distributed evenly in both the cytoplasm and nucleus, suggesting that C31 dominated the intracellular localization of the fusion proteins.
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The 451RFGK454 site is essential for
C31 integrase activityTo determine whether the DAXX binding site, 451RFGK454, on
C31 is involved in the activity of the integrase, pcDNA-C31, pcDNA-C31(Del451–454), pcDNA-C31(R451H) or pcDNA-C31(K454N) was co-transformed with pBCPB into E.coli DH5-competent cells. Twenty-four hours later, pcDNA-C31(R451H) gave rise to a similar number of positive colonies as that of pcDNA-C31, (51.20% ± 1.60% versus 49.98% ± 2.63%). Thus, mutation from Arg451 to His451 in C31 did not affect the integrase activity. In contrast, no white colonies were seen in samples transformed with pcDNA-C31(Del451–454) and pcDNA-C31(K454N), indicating that 451RFGK454 and Lys454 are essential for the activity of C31 integrase.
DAXX inhibits C31-mediated integration
The C31 activity reporter cells PB[EGFP] transfected with pCMVInt gave rise to a recombination rate in the total transfectants of 20.30% ± 1.04% when the transfection efficiency averaged 47.50% ± 3.04%. Co-expression of pDAXX-DsRed but not a control plasmid pDsRed1 significantly reduced the recombination rate by 39.40 ± 0.98% (P < 0.01). On the other hand, co-transfection with pCMTInt and a DAXX-specific duplex small RNA resulted in an increase in GFP-expressing cells of 24.24 ± 0.95% (P < 0.05). However, this increased rate of recombination was not seen in cells co-transfected with a human non-silencing duplex small RNA (82.25 ± 1.05% of the control, P > 0.09, Figure 3 column D). These results indicated that the endogenous DAXX can slightly inhibit the integration efficiency of C31 in HEK293 cells (Figure 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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DAXX is known for its distribution in nucleus domain 10 (ND10) and it relocates to the cytoplasm through nuclear export in response to certain signals (16–18). Consistent with this, we found that punctate DAXX-DsRed fluorescence was mainly localized in the nucleus. Meanwhile, the GFP-
C31 fusion protein was predominantly located in the cytoplasm. The GFP fusion protein possesses the same subcellular distribution as C31 itself, and was localized with an antibody against C31 protein (to be published elsewhere). The predominant cytoplasmic localization of C31 was also consistent with an earlier observation that adding a nuclear localization signal could increase the integration efficiency in mammalian cells (19). The lack of apparent co-localization between DAXX and C31 integrase with fluorescent microscopy in contrast to the high affinity between the two proteins demonstrated by yeast-two-hybrid screening and co-immunoprecipitation may be attributed to the insufficient sensitivity of fluorescence microscopy and/or low amounts of the co-localized proteins. Strong red fluorescence of the DAXX-DsRed fusion protein in the nucleus may mask the green fluorescence of EGFP-C31 fusion protein due to the relatively small amount of intranuclear-localized C31 and vice versa for the cytoplasmic-localized DAXX-DsRed. Indeed, low levels of EGFP fluorescence seemed present in the nuclei of a few cells under microscope although the fluorescence failed to show in microphotographs. This argument is supported by the fact that, although little EGFP-C31 green fluorescence was seen in the nuclei, C31-mediated site-specific integration, which only takes place in the nucleus, was found in a significant percentage of C31 activity reporter cells. Furthermore, a functional interaction between the two proteins was also demonstrated by both overexpression and knockdown DAXX in HEK293 cells. It is especially noteworthy that the C31-mediated integration rate was increased by 24% in DAXX siRNA-treated cells because this suggests the existence of an endogenous interaction between the two proteins. The relatively mild increase in the C31 integration efficiency may be attributed to the extreme abundance of DAXX in the nucleus causing incomplete DAXX knock down by the siRNA. However, it is important to determine whether C31 predominantly localized in the cytoplasm can interfere with the function of DAXX when the latter is translocated to the cytoplasm upon activation by other cellular signals.
The mutational analysis of C31 protein showed that the DAXX binds to 451RFGK454 in the C31. The integrase activity assay further indicated that the tetramer is essential for integrase activity and therefore, binding to this region by DAXX may inhibit the integrase. It is worth pointing out that previous studies have indicated that the catalytic activity of C31 was located between residues 1 and 120 at the N-terminus of the protein, and that the Ser12 is the putative catalytic serine while the putative DNA binding domain is possibly at the C-terminus (20,21). We showed here that 451RFGK454 is crucial for the integration activity of C31. However, the exact role of this region remains to be investigated.
Our yeast-mating test showed that only the construct containing the DAXX 627–740 fragment, but not DAXX 573–627, interacted with C31, suggesting that this 627–740 amino acid region is crucial for the DAXX-C31 interaction. Interestingly, the same domain is also well known for its interaction with other cellular proteins including Fas. To date, more than 20 proteins have been reported to interact with DAXX resulting in a broad range of effects including apoptotic regulation, transcriptional regulation, chromatin remodeling, involvement in signal pathways and regulation of the interaction between virus and host cells (22,23). DAXX was also reported to bind to the integrase of Avian Sarcoma Virus (ASV) (24). Interestingly, we found that DAXX binds to C31 through the same domain as that for binding to ASV integrase. More importantly, the interaction between DAXX and ASV integrase causes suppression of viral replication, which was also reported in human cytomegalovirus-infected cells where DAXX had also been found to interact with a viral protein, pp71, causing inhibition of viral replication (25–27), suggesting that DAXX may play a role in cellular defense against viral infection.
The present study was the first to reveal that a phage integrase, C31, interacts with an important cellular protein, DAXX. The strong binding of C31 to DAXX indicates the importance of understanding potential interactions between the integrases and cellular proteins in mammalian cells since the information gained would aid in developing a gene therapy strategy for site-specific genomic insertion. On the other hand, the inhibitory effect of DAXX on viral proteins across a wide range of species (from phage, avian to human viruses) suggests that DAXX might play a role in maintaining the stability of the genome through preventing extrinsic DNA integration.
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ACKNOWLEDGEMENTS |
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We are very grateful to Michele P. Calos for providing the pCMVInt and pBCBP+ plasmids. We thank PepMetric Technologies for their help in peptide array technique. We also thank Ms.Cathy Campbell for the assistance in preparing this manuscript. This work was supported by the National Basic Research Program of China (973, 2004CB518803). Funding to pay the Open Access publication charges for this article was provided by 973 project (2004CB518803).
Conflict of interest statement. None declared.
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Footnotes |
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*Correspondence may also be addressed to William Jia. Tel: +604 822 0728; Fax: +604 322 0640; Email: wjia{at}interchange.ubc.ca
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