Human RNase H-mediated RNA cleavage from DNA-RNA duplexes is inhibited by 6-deoxythioguanosine incorporation into DNA

Mercaptopurine and thioguanine are anticancer and immunosuppressive agents that exert their primary cytotoxic effects via incorporation of deoxythiogu anosine (dG(s)) into DNA, but the precise mechanism(s) by which this causes cytotoxicity remains unknown. We initially determined that the level of dG (s) incorporation into DNA of human T- and B-lineage leukemia cell lines di d not correlate significantly with the extent of cytotoxicity (IC50), excep t that there was no cytotoxicity in the absence of dG(s) incorporation. To elucidate biological processes perturbed by dG(s) incorporation into DNA, w e chemically synthesized oligodeoxyribonucleotides containing a single dG(s ) (11 mer and 19 mer), which decreased the melting temperature (T-m) of DNA -DNA duplexes without major structural changes, as evidenced by circular di chroism spectra. Using nuclear extracts from human lymphoblastic leukemia c ells (CCRF-CEM, NALM6, and Molt4), we documented that dG(s) incorporation i nto the DNA strand of DNA-RNA heteroduplexes significantly inhibited human RNase H-catalyzed RNA cleavage (80-90% inhibition) and that a similar inhib ition was evident with bacterial RNase H. These data provide the first evid ence that thiopurines inhibit the function of RNase H, indicating that thei r mechanism of cytotoxicity may involve interference with this component of the replication machinery.