Induction of DNA Double-Strand Breaks by Electroporation of Restriction Enzymes into Mammalian Cells

The introduction of restriction endonucleases into mammalian cells in culture provides a unique method for introducing double-strand breaks (DSBs) into the DNA of the host cell. Restriction enzymes recognize, bind, and cleave specific DNA sequences to produce a DNA DSB in the absence of other types of DNA damage. Not only is the DSB the only lesion produced in DNA, but the type of break (i.e., whether it has blunt ends or cohesive ends with 3′- or 5′-overhangs), the nucleotide sequences at the break site, and in certain instances, the precise chromosomal location of the induced break are also known. Once inside a cell, restriction enzymes are extremely efficient inducers of DSBs as measured by pulsed-field gel electrophoresis (1 ) and neutral filter elution (2 ). Consequently, the introduction of restriction enzymes into cells has been used to investigate the biological consequences of DSBs in the genome at the molecular, cytogenetic, and cellular level. At the molecular level, the rejoining of DSBs with known end structure has been investigated (3 ), and although it is not possible to determine the frequency of faithfully repaired DSBs, Phillips and Morgan (4 ) were able to describe unambiguously illegitimate recombination events occurring within the chromosome. At the cellular level, restriction enzyme-induced DSBs lead to mutations (5 ,6 ), chromosome rearrangements (7 –10 ), gene amplification (11 ) and cell killing (5 ,10 ). Interestingly, restriction enzymes do not induce delayed chromosomal instability, suggesting that DSBs are not the signal responsible for initiating this process (12 ).