INDUCTION OF CLEAVAGE IN TOPOISOMERASE-I C-DNA BY TOPOISOMERASE-I ENZYMES FROM CALF THYMUS AND WHEAT-GERM IN THE PRESENCE AND ABSENCE OF CAMPTOTHECIN

In this study, we further examined the sequence selectivity of camptot hecin in mammalian topoisomerase I cDNA from human and Chinese hamster . In the absence of camptothecin, almost all the bases at the 3'-termi nus of cleavage sites are T for calf thymus and wheat germ topolsomera se 1. In addition, wheat germ topoisomerase I exhibits preference for C (or not T) at -3 and for T at -2 position. As for camptothecin-stimu lated cleavage with topoisomerase 1, G (or not T) at + 1 is an additio nal strong preference. This sequence selectivity of camptothecin is si milar to that previously found in SV40 DNA, suggesting that camptothec in preferentially interacts with topolsomerase 1-mediated cleavage sit es where G is the base at the 5'-terminus. These results support the s tacking model of camptothecin (Jaxel et al. (1991) J. Biol. Chem. 266, 20418 - 20423). Comparison of calf thymus and wheat germ topoisomeras e I-mediated cleavage sites in the presence of camptothecin shows that many major cleavage sites are similar. However, the relative intensit ies are often different. One of the differences was attributable to a bias at position - 3 where calf thymus topoisomerase I prefers G and w heat germ topoisomerase I prefers C. This difference may explain the u nique patterns of cleavage sites induced by the two enzymes. Sequencin g analysis of camptothecin-stimulated cleavage sites in the surroundin g regions of point mutations in topolsomerase I cDNA, which were found in camptothecin-resistant cell lines, reveals no direct relationship between DNA cleavage sites in vitro and mutation sites.