DNA Damage Repair Design and Protocols

DNA Damage Repair Design / Protocol

When designing oligos incorporating modified bases suitable for DNA damage/repair studies, it is important to properly match the type of lesion under study and the analytical method chosen for detection.

The standard in vitro assay for NER is a reconstitution of this repair system in cell-free extract using six recombinantly expressed NER factors (RPA, XPA, XPC, TFIIH, XPG, and XPF)(9) and a synthetic oligo duplex as template, modified with an adduct known to induce NER (10).

The standard in vitro assay for BER is a reconstitution of this repair system in whole cell extracts using a synthetic oligo duplex as template, containing a modified base (e.g., 8-oxo-dG) known to induce BER (11). It is also possible to experimentally monitor BER in vivo (12).

For studying UV-light induced DNA damage (formation of cyclobutane pyrimidine dimers (CPDs) or 6,4-photoproducts) in particular genomic regions, PCR-based techniques typically are the analytical method of choice, most commonly ligation-mediated PCR (LMPCR) (13). However, PCR-based methods are not suitable for telomeric regions, because telomeres are composed of thousands of copies of the short tandem repeat 5′TTAGGG/5′CCCTAA, and thus have no unique PCR priming sites. So, for telomeric regions, immunoprecipitation of DNA damage (IPoD) is used (14).

References

(1) Lodish, H., Berk, A., Matsudaira, P., Kaiser, C.A., Krieger, M., Scott, M.P., Zipursky, S.L., Darnell, J. (2004). In Molecular Biology of the Cell 5th ed., WH Freeman, New York, NY, 963.
(2) Lindahl, T. (1993) “Instability and decay of the primary structure of DNA”, Nature 362: 709-715.
(3) Nilsen, H., Krokan, H.E. (2001) “Base excision repair in a network of defence and tolerance”, Carcinogenesis 22: 987-998.
(4) de Laat, W.L, Jaspers, N.G.J., Hoeijmakers, J.H.J. (1999) “Molecular mechanism of nucleotide excision repair”, Genes & Development 13: 768-785.
(5) Iyer R., Pluciennik A., Burdett V., Modrich P. (2006). "DNA mismatch repair: functions and mechanisms". Chem Rev 106: 302–23.
(6) Sancar A. (2003). “Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors”, Chem Rev 103:2203–37.
(7) Shikazono, N., Pearson, C., O’Neill, P., Thacker, J. (2006) “The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA”, Nucleic Acids Res. 34: 3722-3730.
(8) Margolin, Y., Shafirovich, V., Geacintov, N.E., DeMott, M.S., Dedon, P.C. (2008) “DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-Electron Oxidants”, J. Biol. Chem. 283: 35569-35578.
(9) Reardon, J.T., Sancar, A. Recognition and repair of the cyclobutane thymine dimer, a major cause of skin cancers, by the human excision nuclease. Genes Dev (2003), 17: 2359-2551.
(10) Prakash, S., Prakash, L. Nucleotide excision repair in yeast. Mutat. Res. (2000), 451: 13-24.
(11) Asagoshi, K., Tano, K., Chastain, P.D., et al. FEN1 Functions in Long Patch Base Excision Repair Under Conditions of Oxidative Stress in Vertebrate Cells. Mol Cancer Res (2010), 8: 204-215.
(12) Sundaresakumar, P. Use of novel assays to measure in vivo base excision DNA repair. M.S. Thesis, San Jose State Univ, 2009, 105 pages, Pub # 1470953. (13) Tornaletti, S., Pfeifer G.P. Ligation-Mediated PCR for Analysis of UV damage. In: Pfeifer, G.P., ed., Technologies for detection of DNA damage and mutations. New York: Plenum Press, 1996, pp. 199-209.
(14) Rochette, P.J., Brash, D.E. Human Telomeres Are Hypersensitive to UV-Induced DNA Damage and Refractory to Repair. PLoS Genetics (2010), 6(4): e1000926.