Dr. Mikhail (Misha) Kolot
Ph.D.: Moscow University, former USSR, 1985
Phone: (Phone) +972-3-640-9823
(Fax) +972-3-640-6834
E-mail: kolott@post.tau.ac.il
Room#: Sherman Building, Room 607
Member's portrait

Research Interests

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

The main research interest of our lab is to understand the mechanism of the site specific recombination system of bacteriophage HK022 and its application for gene manipulations in human cells and in zebra fish cells.  

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). 

fig1
Figure 1. Integration and excision of phage HK022 and lambda. As a result of crossover between attP (PoPג€™) and attB (BoBג€™) a prophage is formed which is flanked by the recombination sites attL (BoPג€™) and attR (PoBג€™).


We have cloned and expressed the Int recombinase of phage HK022's in human cells, in plants, cyanobacterium (Anabaena) and in zebra fish 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, Harel-Levi et al., 2008, Gottfried et al. , 2005 Melnikov et al., 2009 and unpublished, Fig. 2).


fig2
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.

fig3

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 replace the Neo R gene with the HGPRT + cDNA gene. E. Cured HGPRT + cell line.

The introduction of the Int system into zebra fish (Fig. 4) is for the purpose of spatial inactivation of the pineal gland at various developmental stages in order to study the role of the pineal gland in the circadian cycle. This work is in collaboration with the lab of Dr. Yoav Gothilf at the Neurobiochemistry Dept.

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 and gene therapy.
fig4
Figure 4. A, Developing zebra fish embryo injected with a red-fluorescent substrate plasmid for site-specific recombination in the presence of Int. Recombination causes green fluorescence B. using a red filter showing the presence of the substrate; C. using a green filter showing recombination activity; using D. using both filters. Yellow spots show combined red and green fluorescence.

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-Dֳ¼by, 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 ֿƒ 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., Drֳ¶ge 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., Kolot, M. (2009) Site-specific recombination  in cyanobacterium Anabaen PCC7120 catalyzed by the integrase of bacteriophage HK022. J. Bacteriol. 191:4458-4464. (pdf)
  25. Malchin, N., Molotsky, T., Borovok, I., Voziyanov, Y.,  Kotlyar A.B., Yagil, E. and Kolot, M. High Efficiency of a Sequential Recombinase-Mediated Cassette Exchange Reaction in E. coli. Submitted for publication.

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