Research Interests

Mechanism of the lambdoid site-specific recombination system and its use for gene insertions in the eukaryotic genome

The bacterial viruses (bacteriophages) Lambda and its relative HK022 can insert their genomes at specific sites on the chromosome of their host, Escherichia coli. This is accomplished by a process known as site-specific recombination. The reaction results from a crossover between a specific site (attP) on the viral DNA and a site (attB) on the bacterial DNA. The reaction is catalyzed by the Integrase (Int) protein that is encoded by the virus. In addition, there are two accessory proteins (IHF and Xis) that participate in this process. Int, IHF and Xis bind to specific sites on their DNA substrates and form a complex known as the intasome. This site-specific recombination reaction can also be performed in vitro.

In our laboratory we study basic questions to elucidate the mechanism of this site-specific recombination reaction and its evolutionary aspects. We also develop the system to be used as a biotechnological tool to insert or excise desired DNA fragments in the genome of mammals and of plants.

In the basic approach we study how Int and the accessory proteins interact with each other and with their DNA substrates in the formation of the intasome. We do crosslinking experiments between the various components of the intasome and by a partial proteolysis and amino acid determination we elucidate the amino acids that are responsible for the protein-protein and protein-DNA interactions. These experiments teach us the mechanism of the site-specific recombination reaction.
In the biotechnological approach we have cloned and expressed the Int protein in mammalian and in plant cells and have shown that, when supplied with the proper DNA att sites, Int is active in the eukaryotic milieu. We develop this system to insert and replace desired DNA sequences at specific sites of the eukaryotic genome for the purpose of future gene therapy and plant improvement

Selected Publications

1. Yagil, E., S. Dolev, J. Oberto, N. Kislev, N. Ramaiah and R.Weisberg. (1989). The determinants of site-specific recombination in the lambdoid coliphage HK022: An evolutionary change in specificity. J. Mol. Biol. 207:695-717.
2. Kolot M., and E. Yagil. (1994). Position and direction of strand exchange in bacteriophage HK022 integration. Molec. Gen. Genet. 245:623-637.
3. Nunes-Düby, S. E., R. S. Tirumalai, L. Dorgai, E. Yagil, R. A. Weisberg and A. Landy (1994). Lambda integrase cleaves DNA in cis. EMBO J. 13:4421-4430.
4. Yagil, E., L. Dorgai, and R.A. Weisberg (1995). Identifying determinants of recombination specificity: Construction and characterization of chimeric bacteriophage integrases. J. Molec. Biol. 252:163-177 (pdf).
5. Dorgai, L., E. Yagil, and R.A. Weisberg (1995). Identifying determinants of recombination specificity: Construction and characterization of mutant bacteriophage integrases. J. Molec. Biol. 252:178-188 (pdf).
6. Kolot, M. P. Gottfried,  and E. Yagil (1996). A second site-specific recombination event in the lambdoid bacteriophage HK022. Molec. Gen. Genet. 253:362-369.
7. Kolot, M., Silberstein, N. and E. Yagil. (1999) Site specific recombination in mammalian cells expressing the Int recombinase of bacteriophage HK022. Molec. Biol. Reprots 26:207-213.
8. Gottfried, P., Yagil, E. and M. Kolot. (2000) Core-binding specificity of bacteriophage integrases. Molec. Gen. Genet. 263:619-624.
9. Gottfried, P., M. Kolot, and E. Yagil. (2001) The effect of mutations in the Xis binding sites on site-specific recombination in coliphage HK022. Molec. Gen. Genet. 266:584-590 (pdf).
10. Aguena, M., E. Yagil, and B. Spira. (2002) Transcriptional analysis of the pst operon of Escherichia coli . Molec. Gen. Genomics 268:518-524 (pdf).
11. 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).
12. Rutkai, E., Dorgai, L., Sirot, R., Yagil, E. and R. Weisberg (2003) Characterization of secondary attachment sites used by phage l, and a role for such sites in changing phage insertion specificity. . J. Molec. Biol. 329:983-996 (pdf).
13. 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).
14. 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).
15. Taschner, N. P., Yagil, E. and Spria, B. (2004) A differential effect of sS on the expression of the PHO regulon genes of Escherichia coli. Microbiology, 150: 2985-2992 (pdf).
16. 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).
17. 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).
18. Pasternak-Tashner, N., Yagil, E., and Spria B. (2006) The effect of IHF on σ S selectivity of the phoA and pst promoters of Escherichia coli . Archives of Micrbiology:185:234-237 (pdf).

19. Yemini,M., Levi, Y., Yagil, E. And Rsihpon, J. (2007) Specific electrochemical phage sensing for Bacillus subtilis and Mycobacterium smegmatis . Bioelectrochemistry 70:180-184 (pdf).

20. 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).

21. 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).

22. 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).

23. 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).

24. 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