Research Interests

The main research interest of our lab is understanding the mechanism of the HK022 bacteriophage site specific recombination system and its use for gene manipulations in eukaryotic cells.

Site-specific recombinases mediate precise, conservative recombination between two short DNA recognition target sites. Four types of DNA rearrangement can result from site-specific mediated recombination: integration, excision, resolution and inversion. During the last decade such systems have become widely used as a powerful tool in cell line and organism genome engineering.

The site-specific recombination mechanism of coliphage HK022 is similar to that of its well-known relative coliphage lambda (Fig. 1). The bacterial site ( attB ) is 21 bp long, while the phage site ( attP ) is over 200 bp long, the latter carrying binding sites for accessory proteins required for the phage-encoded Int recombinase to catalyze the recombination reactions. The integration reaction ( attB x attP ) results in the insertion of the phage genome into the host chromosome. As a result, the integrated prophage is flanked by the newly structured recombinant attL and attR sites, which serve as substrate for the reverse excision reaction (Fig. 1). 

Figure 1. Integration and excision of phage HK022 and lambda. As a result of crossover between attP (PoP’) and attB (BoB’) a prophage is formed which is flanked by the recombination sites attL (BoP’) and attR (PoB’).

We have cloned and expressed the Int recombinase of phage HK022's in human cells, in plants and in zebrafish and found that when supplied with the requisite att site pairs, Int can catalyze the integration ( attP x attB ) and excision ( attL x attR ) reactions in these organisms (Kolot et al. , 2003, Gottfried et al. , 2005 and unpublished, Fig. 2).

Figure 2. Int promotes the site specific recombination in human cells. The structure of the plasmids used as extrachromosomal substrates (numbered circles) and the expected products of the Int-catalyzed recombination. Recombination between the two att sites evicts the transcription terminator (STOP) in cis configuration, (A, C) or in the trans configuration, connecting two substrates (B, D). As a result the CMV promoter allows the expression of the GFP gene. The inserts show dot plots of the FACS analyses in the absence (bottom) and the presence (top) of the Int-expressing plasmid.

Using the Recombination-Mediated Casette Exchange (RMCE) technology we are currently investigating, as a model, if the Int recombinase can cure the human hereditary Lesch-Nyhan syndrome that is caused by the deficiency of the HGPRT enzyme. Figure 3 outlines the strategy that we use.

Figure 3. Using the RMCE technology to cure the HGPRT hereditary defect. A. Plasmid used for the construction of the RMCE platform with two attR sites, to be introduced by Hyg R selection into the HGPRT mutated cell line genome (B) as a result of homologous recombination via the H1 and H2 regions . C. RMCE platform cell line that carries the two attR sites and the Neo R gene. D. Plasmid to to replace the Neo R gene with the HGPRT + cDNA gene. E. Cured HGPRT + cell line.

We expect that our research will lead to the development of efficient and universal genome manipulation technologies based on the Int recombinase of HK022 . These technologies will have a significant impact on future biomedical research.

Selected Publications

 

List of publications (recent years only)

1. Kolot, M. and E. Yagil. (2003) Determinants that target the integrase of phage HK022 into the mammalian nucleus. J. Molec. Biol. 325:629-635 (pdf).
2. Kolot, M., Meroz, A. and E. Yagil. (2003) Site-specific recombination in human cells catalyzed by the wild type integrase protein of coliphage HK022. Biotechnology and Bioengineering84:56-60 (pdf).
3. Gottfried, P., Silberstein, N., Yagil, E and Kolot, M. (2003) Activity of coliphage HK022 excisionase (Xis) in the absence of DNA binding. FEBS Letters 545:133-138 (pdf).
4. Gottfried, P., Kolot, M., Silberstein, N. and Yagil E. (2004) Protein-protein interaction between monomers of coliphage HK022 excisionase. FEBS Letters, 577:17-20 (pdf).
5. Gottfried, P., Lotan, O., Kolot, M., Maslenin, L., Bendov, R., Gorovits, R., Yesodi, V., Yagil, E. and Rosner, A. (2005) Site-specific recombination in Arabidopsis plants promoted by the integrase protein of coliphage HK022. Plant Molecular Biology 57:435-444 (pdf).

6. Harel-Levi G., Goltsman J., Tuby CN., Yagil E. and Kolot M. (2008) Human genomic site-specific recombination catalyzed by coliphage HK022 integrase. J Biotechnol. 134 : 46-54 (pdf).

7. Kolot M., Gorovits R., Silberstein N., Fichtman B. and Yagil E. (2008) Phosphorylation of the integrase protein of coliphage HK022. Virology. Virology 375:383-390 (pdf).

8. Malchin N., Molotsky T., Yagil E., Kotlyar A B. and Kolot M. (2008) Molecular analysis of a recombinase-mediated cassette exchange reactions in Escherichia coli catalyzed by integrase of coliphage HK022. Res. Microbiol. 159:663-670 (pdf).

9. Malchin N., Goltsman J., Gorovits R., Bao Q., Dröge P., Yagil, E. and Kolot M. (2009) Optimization of coliphage HK022 integrase activity in human cell. Gene 437:9-13 (pdf).

10. Melnikov, O., Zaritsky, A., Zarka, A., Boussiba, S., Yagil, E. and Kolot M. (2009) Site-specific recombination in the cyanobacterium Anabaena PCC7120 catalyzed by the integrase of bacteriophage HK022 . J. Bacteriol. In Press