Comparative utilization of bromodeoxyuridine and iododeoxyuridine triphosphates for mammalian DNA replication in vitro

Abstract

Relative utilization, of dThd, BrdUrd, and IdUrd as exogenous precursors of DNA in vivo was compared with utilization of the respective triphosphates in vitro. To measure relative utilization, exponentially growing P-815 or CHO cells were incubated with different mixtures of dThd/BrdUrd or dThd/IdUrd in the presence of FdUrd and dCyd, and Brij-58 lysed cells with mixtures of dTTP/BrdUTP or dTTP/IdUTP. The degree of substitution of dTMP by BrdUMP or IdUMP in DNA newly synthesized in vivo or in vitro was determined by isopycnic centrifugation. BrdUrd was utilized by intact cells with equal efficiency as dThd, while IdUrd was less efficiently incorporated when the total concentration of dThd and the tested analogue was 3.10(-5) M. During semiconservative DNA replication in vitro, however, dTTP, BrdUTP and IdUTP were utilized with equal efficiency. Thus, we conclude that observed preference for dThd by intact cells incubated with dThd/IdUrd mixtures is related to characteristics of precursor metabolism and not to different affinities of the replication complex for the respective precursors. dUTP was rapidly dephosphorylated in vitro and no incorporation into DNA was detected.