Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks , alkylations , or x - ray damage ( chromosomes / repair / DNA damage )

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plusGiemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions. When cells are exposed to ultraviolet radiation (1) or to chemical mutagenic carcinogens (2), one of the most readily observable effects is the induction of chromosomal exchanges between sister chromatids. If the DNA is allowed to replicate in the presence of the thymidine analog 5-bromodeoxyuridine (BrdUrd), in which the methyl group of the thymidine is replaced with the heavier atom bromine, then, because of the semiconservative replication of DNA, each sister chromatid contains one original light polynucleotide strand and one new heavy strand containing BrdUrd. If such a chromatid replicates again in the presence of BrdUrd, each polynucleotide strand separates from its complementary strand and is now paired with a new heavy strand. This results in chromosomes in which one sister chromatid is bifilarly labeled with BrdUrd, and the other sister chromatid is only unifilarly labeled. The two sister chromatids are now chemically different from one another and can be made to stain differentially (3). The chromatid containing more BrdUrd always stains lighter than its sister. Any The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. sister chromatid exchange (SCE) can now be seen clearly with great resolution (4). In fact, the induction of SCEs visible in such chromosomes has constituted one of the most sensitive mammalian tests for the effects of mutagenic carcinogens (5). Because cells must replicate at least twice before an SCE becomes visible, any given SCE could have been induced at either of the S periods before the cells reach the metaphase at which the exchanges are scored (1). In cells exposed to chemical agents, this has raised questions of how long any lesion, or adduct, remains unrepaired in the DNA and therefore remains capable of producing an SCE. In previous attempts to determine in which cell cycle an SCE was actually formed, cells often were cultured for three rounds of replication in the presence of BrdUrd, which led to threeway differential staining of the chromosomes (6-8). This method frequently is subjective because of the vagaries in stain intensity brought about by BrdUrd depletion, which leads to varying amounts of BrdUrd being incorporated in each round of replication, as well as by other complications of the fluorescence-plus-Giemsa (FPG) staining technique. In Chinese hamster ovary (CHO) cells that have already undergone two rounds of DNA replication, we have now induced chromosomes to undergo endoreduplication at high frequency (-65%) by treating the cells with rotenone according to the method of Matsumoto and Ohta (9, 10). If the replication cycle that results in endoreduplication takes place in the absence of BrdUrd, then the endoreduplicated chromosome pairs consist of one chromosome in which both chromatids are unifilarly substituted and will stain lightly by the FPG technique, and one chromosome that contains a darkly stained sister chromatid with unsubstituted DNA and a lightly stained chromatid with unifilarly substituted DNA (Fig. 1). A similar staining pattern is obtained even if BrdUrd is present in the replication cycle that results in endoreduplication, but in this case the light chromatid will be bifilarly substituted and the dark chromatid will be unifilarly substituted. Because of the topological constraints that lead to the newly replicated polynucleotide strand always segregating to the outside of a pair of sister chromatids (4, 11-13), in either case the endoreduplicated pair then consists of a chromosome with a light chromatid and its light sister chromatid lying next to a dark chromatid from the chromosome containing a dark chromatid and a light sister chromatid (Fig. 2). Thus the order of the chromatids in such an endoreduplicated pair of chromosomes is light-dark next to light-light, with the dark chromatid always in the center. An SCE will disrupt this pattern. Furthermore, in such cells, by using the criteria of Brewen and Peacock (14) that were established for SCEs Abbreviations: SCE, sister chromatid exchange; FPG, fluorescenceplus-Giemsa; CHO, Chinese hamster ovary; MMC, mitomycin C; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; BrdUrd, 5-bromodeoxyuridine. *To whom reprint requests should be addressed. e-mail: shelly@ radlab.ucsf.edu.