Abstract
DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA repair in Dictyostelium. Genes required for repair of DNA damage can be identified and analyzed by comparing the ability of control or mutant cells to survive exposure to genotoxic agents that induce different types of DNA lesion. We also describe assays that assess the presence of markers for DNA repair within chromatin either in the form of posttranslational modification of proteins at sites of damage or the recruitment of repair factors to DNA lesions. Finally, we also describe more direct assays to assess repair of DNA double-strand breaks by either homologous recombination or non-homologous end joining.
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References
Deering RA (1968) Dictyostelium discoideum: a gamma-ray resistant organism. Science 162:1289–1290
Zhang XY, Langenick J, Traynor D, Babu MM, Kay RR, Patel KJ (2009) Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the extreme resistance to cisplatin in Dictyostelium discoideum. PLoS Genet 5:e1000645
Hudson JJ, Hsu DW, Guo K, Zhukovskaya N, Liu PH, Williams JG, Pears CJ, Lakin ND (2005) DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum. Curr Biol 15:1880–1885
Block WD, Lees-Miller SP (2005) Putative homologues of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and other components of the non-homologous end joining machinery in Dictyostelium discoideum. DNA Repair 4:1061–1065
Hsu DW, Kiely R, Couto CA, Wang HY, Hudson JJ, Borer C, Pears CJ, Lakin ND (2011) DNA double-strand break repair pathway choice in Dictyostelium. J Cell Sci 124:1655–1663
Hsu DW, Gaudet P, Hudson JJ, Pears CJ, Lakin ND (2006) DNA damage signaling and repair in Dictyostelium discoideum. Cell Cycle 5:702–708
Muramoto T, Chubb JR (2008) Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint. Development 135:1647–1657
Kofler B, Wallraff E, Herzog H, Schneider R, Auer B, Schweiger M (1993) Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum. Biochem J 293: 275–281
Rickwood D, Osman MS (1979) Characterisation of poly(ADP-Rib) polymerase activity in nuclei from the slime mould Dictyostelium discoideum. Mol Cell Biochem 27:79–84
Citarelli M, Teotia S, Lamb RS (2010) Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes. BMC Evol Biol 10:308
Couto CA, Wang HY, Green JC, Kiely R, Siddaway R, Borer C, Pears CJ, Lakin ND (2011) PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks. J Cell Biol 194:367–375
Pears CJ, Couto CA, Wang HY, Borer C, Kiely R, Lakin ND (2012) The role of ADP-ribosylation in regulating DNA double-strand break repair. Cell Cycle 11:48–56
Li G, Alexander H, Schneider N, Alexander S (2000) Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146:2219–2227
Lee SK, Yu SL, Alexander H, Alexander S (1998) A mutation in repB, the Dictyostelium homolog of the human xeroderma pigmentosum B gene, has increased sensitivity to UV-light but normal morphogenesis. Biochim Biophys Acta 1399:161–172
Min J, Sridevi P, Alexander S, Alexander H (2006) Sensitive cell viability assay for use in drug screens and for studying the mechanism of action of drugs in Dictyostelium discoideum. Biotechniques 41:591–595
Drouet J, Delteil C, Lefrancois J, Concannon P, Salles B, Calsou P (2005) DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks. J Biol Chem 280:7060–7069
Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR (2004) A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res 32:e143
Lin HH, Khosla M, Huang HJ, Hsu DW, Michaelis C, Weeks G, Pears C (2004) A homologue of Cdk8 is required for spore cell differentiation in Dictyostelium. Dev Biol 271:49–58
Greene DM, Bloomfield G, Skelton J, Ivens A, Pears CJ (2011) Targets downstream of Cdk8 in Dictyostelium development. BMC Dev Biol 11:2
Kuspa A, Loomis WF (1992) Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A 89:8803–8807
Demsar IH, Cotter DA (1981) Physiological effects of ultraviolet light on Dictyostelium discoideum spore germination. Photochem Photobiol 34:455–460
Maeda M, Kuwayama H (2000) A diffusible factor involved in MAP-kinase ERK2-regulated development of Dictyostelium. Dev Growth Differ 42:275–284
Garcia MX, Alexander H, Mahadeo D, Cotter DA, Alexander S (2003) The Dictyostelium discoideum prespore-specific catalase B functions to control late development and to protect spore viability. Biochim Biophys Acta 1641:55–64
Gaudet P, Tsang A (1999) Regulation of the ribonucleotide reductase small subunit gene by DNA-damaging agents in Dictyostelium discoideum. Nucleic Acids Res 27:3042–3048
Podgorski G, Deering RA (1980) Effect of methyl methanesulfonate on survival of radiation-sensitive strains of Dictyostelium discoideum. Mutat Res 73:415–418
Bronner CE, Welker DL, Deering RA (1992) Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents. Mutat Res 274:187–200
Freeland TM, Guyer RB, Ling AZ, Deering RA (1996) Apurinic/apyrimidinic (AP) endonuclease from Dictyostelium discoideum: cloning, nucleotide sequence and induction by sublethal levels of DNA damaging agents. Nucleic Acids Res 24:1950–1953
Ling AZ, Guyer RB, Deering RA (2001) Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter. Environ Mol Mutagen 38:244–247
Deering RA, Guyer RB, Stevens L, Watson-Thais TE (1996) Some repair-deficient mutants of Dictyostelium discoideum display enhanced susceptibilities to bleomycin. Antimicrob Agents Chemother 40:464–467
Van Driessche N, Alexander H, Min J, Kuspa A, Alexander S, Shaulsky G (2007) Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets. Proc Natl Acad Sci USA 104: 15406–15411
Yu SL, Lee SK, Alexander H, Alexander S (1998) Rapid changes of nucleotide excision repair gene expression following UV-irradiation and cisplatin treatment of Dictyostelium discoideum. Nucleic Acids Res 26:3397–3403
Hurley DL, Skantar AM, Deering RA (1989) Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum. Mutat Res 217:25–32
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Couto, AM.C., Lakin, N.D., Pears, C.J. (2013). Investigation of DNA Repair Pathway Activity. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology, vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_16
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DOI: https://doi.org/10.1007/978-1-62703-302-2_16
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