Analysis of recombinational repair of DNA double-strand breaks in mammalian cells with I-SceI nuclease.
Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis. DSBs are also induced by chemicals and radiation. I-SceI endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-SceI is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes. As such, I-SceI can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair. DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing. The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed. Nonselective assays detect both HR and NHEJ events. This chapter focuses on the systems for delivering I-SceI nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair.
Nickoloff
Jac A
JA
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, USA.
Brenneman
Mark A
MA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D005785:Gene Conversion; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors; D008322:Mammals; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection / Q000379:methods
D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific