Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.

Fluorescence in situ hybridization and Giemsa staining of metaphase chromosomes were used to determine the relative frequencies of symmetric exchange aberrations (translocations) and asymmetric exchange aberrations (rings, dicentrics, and polycentrics) after exposure of human lymphoblastoid cells to restriction enzymes or X-rays. The yield of symmetric exchanges was determined with the use of chromosome-specific probes for human chromosomes 2 or 4, which were hybridized to metaphase chromosomes from cells exposed to the enzymes PvuII, SacI, or XbaI or 3 or 5 Gy of X-rays. The yield of asymmetric exchanges was determined in Giemsa-stained metaphase chromosomes from the same enzyme-treated or irradiated cell population. About 1.5- to 3-fold more symmetric than asymmetric exchanges were induced after restriction enzyme treatment. However, after X-ray treatment the yield of dicentrics relative to the yield of reciprocal translocations was close to the expected 1:1 ratio.

The potential interaction between restriction enzyme-induced double-strand breaks (dsb) and X-ray-induced lesions in the formation of chromosomal aberrations was investigated in Chinese hamster ovary cells. Either Alu I, which induces blunt-end dsb, or Sau 3AI, which induces cohesive-end dsb, was electroporated into cells, which were irradiated with 2 Gy of X-rays immediately or 15, 30, 60, 120, or 180 min after electroporation. A significant increase in Alu I-induced chromosomal aberrations was observed when cells were irradiated with 0, 15, 30, or 60 min after enzyme exposure, but only additive effects were found when cells were irradiated 120 or 180 min after enzyme exposure. In one of three experiments, cells exposed to Sau 3AI showed a large increase in aberrations when X-irradiated 0 or 15 min after Sau 3AI exposure, and no increase at any time-points thereafter. These results indicate that restriction enzyme-induced dsb can interact with X-ray-induced lesions, resulting in a synergistic increase in chromosomal aberration formation. Furthermore, this interaction depends on both the type of dsb and the time between enzyme and X-ray exposure.

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