DNA end-joining driven by microhomologies catalyzed by nuclear extracts
-
Francisco Boán
Abstract
In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.
References
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). Current Protocols in Molecular Biology (New York, USA: Wiley Interscience).Search in Google Scholar
Bartkova, J., Horejsi, Z., Koed, K., Kramer, A., Tort, F., Zieger, K., Guldberg, P., Sehested, M., Nesland, J.M., Lukas, C., et al. (2005). DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature434, 864–870.10.1038/nature03482Search in Google Scholar
Baumann, P. and West, S.C. (1998). DNA end-joining catalyzed by human cell-free extracts. Proc. Natl. Acad. Sci. USA95, 14066–14070.10.1073/pnas.95.24.14066Search in Google Scholar
Blanco, M.G., Boán, F., and Gómez-Márquez, J. (2004). A paradox in the in vitro end-joining assays. J. Biol. Chem.279, 26797–26801.10.1074/jbc.M402832200Search in Google Scholar
Blanco, M.G., Boán, F., Barros, P., Castaño, J.G., and Gómez-Márquez, J. (2005). Generation of double-strand breaks by two independent enzymatic activities in nuclear extracts. J. Mol. Biol.351, 995–1006.10.1016/j.jmb.2005.06.065Search in Google Scholar
Boán, F., González, A.I., Rodríguez, J.M., and Gómez-Márquez, J. (1997). Molecular characterization of a new human minisatellite that is able to form single-stranded loops in vitro and is recognized by nuclear proteins. FEBS Lett.418, 251–257.10.1016/S0014-5793(97)01389-6Search in Google Scholar
Boán, F., Rodríguez, J.M., and Gómez-Márquez, J. (1998). A non-hypervariable human minisatellite strongly stimulates in vitro intramolecular homologous recombination. J. Mol. Biol.278, 499–505.10.1006/jmbi.1998.1714Search in Google Scholar PubMed
Boán, F., Rodríguez, J.M., Mouriño, S., Blanco, M.G., Viñas, A., Sánchez, L., and Gómez-Márquez, J. (2002). Recombination analysis of the human minisatellite MsH42 suggests the existence of two distinct pathways for initiation and resolution of recombination at MsH42 in rat testes nuclear extracts. Biochemistry41, 2166–2176.10.1021/bi015780iSearch in Google Scholar PubMed
Bois, P. and Jeffreys, A.J. (1999). Minisatellite instability and germline mutation. Cell. Mol. Life Sci.55, 1636–1648.10.1007/s000180050402Search in Google Scholar PubMed
Boulton, S.J. and Jackson, S.P. (1996). Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J.15, 5093–5103.10.1002/j.1460-2075.1996.tb00890.xSearch in Google Scholar
Buard, J., Shone, A.C., and Jeffreys, A.J. (2000). Meiotic recombination and flanking marker exchange at the highly unstable human minisatellite CEB1 (D2S90). Am. J. Hum. Genet.67, 333–344.10.1086/303015Search in Google Scholar PubMed PubMed Central
Elliott, B. and Jasin, M. (2002). Double-strand breaks and translocation in cancer. Cell. Mol. Life Sci.59, 373–385.10.1007/s00018-002-8429-3Search in Google Scholar
Glazer, P.M., Sarkar, S.N., Chisholm, G.E., and Summers, W.C. (1987) DNA mismatch repair detected in human cell extracts. Mol. Cell. Biol.7, 218–224.Search in Google Scholar
Göttlich, B., Reichenberger, S., Feldmann, E., and Pfeiffer, P. (1998). Rejoining of double-strand breaks in vitro by single-strand annealing. Eur. J. Biochem.258, 387–395.10.1046/j.1432-1327.1998.2580387.xSearch in Google Scholar
Haber, J.E. (2000). Partners and pathways repairing a double-strand break. Trends Genet.16, 259–264.10.1016/S0168-9525(00)02022-9Search in Google Scholar
Jeffreys, A.J., Allen, M.J., Armour, J.A.L., Collick, A., Dubrova, Y., Fretwell, N., Guram, T., Jobling, M., May, C.A., Neil, D.L., and Neumann, R. (1995). Mutation processes at human minisatellites. Electrophoresis16, 1577–1585.10.1002/elps.11501601261Search in Google Scholar
Kanaar, R., Hoeijmakers, J.H.K., and van Gent, D.C. (1998). Molecular mechanisms of DNA double-strand break repair. Trends Cell Biol.8, 483–489.10.1016/S0962-8924(98)01383-XSearch in Google Scholar
Karran, P. (2000). DNA double strand break in mammalian cells. Curr. Opin. Genet. Dev.10, 144–150.10.1016/S0959-437X(00)00069-1Search in Google Scholar
Lengauer, C., Kinzler, K.W., and Vogelstein, B. (1998). Genetic instabilities in human cancer. Nature396, 643–649.10.1038/25292Search in Google Scholar PubMed
Liang, L., Deng, L., Chen, Y., Li, G.C., Shao, C. and Tischfield, J.A. (2005). Modulation of DNA end joining by nuclear proteins. J. Biol. Chem.280, 31442–31449.10.1074/jbc.M503776200Search in Google Scholar PubMed
Lieber, M.R., Ma, Y., Pannicke, U., and Schwarz, K. (2003). Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell. Biol.4, 712–720.10.1038/nrm1202Search in Google Scholar PubMed
Mason, R.M., Thacker, J., and Fairman, M.P. (1996). The joining of non-complementary DNA double-strand breaks by mammalian extracts. Nucleic Acids Res.24, 4946–4953.10.1093/nar/24.24.4946Search in Google Scholar PubMed PubMed Central
Pâques, F. and Haber, J.E. (1999). Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev.63, 349–404.10.1128/MMBR.63.2.349-404.1999Search in Google Scholar
Pospiech, H., Rytkönen, A.K., and Syväoja, J.E. (2001). The role of DNA polymerase activity in human non-homologous end joining. Nucleic Acids Res.29, 3277–3288.10.1093/nar/29.15.3277Search in Google Scholar
Sambrook, J. and Russell, D.W. (2001). Molecular cloning: a laboratory manual (Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press).Search in Google Scholar
Singleton, B.K. and Jeggo, P.A. (1999). Double-strand break repair and V(D)J recombination. In: DNA Recombination and Repair, P.J. Smith and C.J. Jones, eds. (Oxford, UK: Oxford University Press), pp. 16–37.Search in Google Scholar
Smith, K.N. and Nicolas, A. (1998). Recombination at work for meiosis. Curr. Opin. Genet. Dev.8, 200–211.10.1016/S0959-437X(98)80142-1Search in Google Scholar
Takata, M., Sasaki, M.S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H. Yamaguchi-Iwai, Y., Shinohara, A., and Takeda, S. (1998). Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J.17, 5497–5508.10.1093/emboj/17.18.5497Search in Google Scholar PubMed PubMed Central
Vergnaud, G. and Denoeud, F. (2000). Minisatellites: mutability and genome architecture. Genome Res.10, 899–907.10.1101/gr.10.7.899Search in Google Scholar PubMed
©2006 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Striated domains: self-organizing ordered assemblies of transmembrane α-helical peptides and lipids in bilayers
- Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors
- Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA
- Spontaneous DNA-DNA interaction of homologous duplexes and factors affecting the result of heteroduplex formation
- DNA end-joining driven by microhomologies catalyzed by nuclear extracts
- A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor
- The zinc finger protein ZNF297B interacts with BDP1, a subunit of TFIIIB
- Pressure- and temperature-induced unfolding studies: thermodynamics of core hydrophobicity and packing of ribonuclease A
- Truncated PrPc in mammalian brain: interspecies variation and location in membrane rafts
- Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization
- p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho
- Human plasma adenosine deaminase 2 is secreted by activated monocytes
- Inhibition of mRNA deadenylation and degradation by different types of cell stress
- Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19INK4d
- Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8
Articles in the same Issue
- Striated domains: self-organizing ordered assemblies of transmembrane α-helical peptides and lipids in bilayers
- Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors
- Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA
- Spontaneous DNA-DNA interaction of homologous duplexes and factors affecting the result of heteroduplex formation
- DNA end-joining driven by microhomologies catalyzed by nuclear extracts
- A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor
- The zinc finger protein ZNF297B interacts with BDP1, a subunit of TFIIIB
- Pressure- and temperature-induced unfolding studies: thermodynamics of core hydrophobicity and packing of ribonuclease A
- Truncated PrPc in mammalian brain: interspecies variation and location in membrane rafts
- Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization
- p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho
- Human plasma adenosine deaminase 2 is secreted by activated monocytes
- Inhibition of mRNA deadenylation and degradation by different types of cell stress
- Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19INK4d
- Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8