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

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.

Semiconservative DNA replication in vitro. I. Properties of two systems derived from mouse P-815 cells by permeabilization or lysis with Brij-58.

To study DNA replication in vitro, mouse P-815 cells were either permeabilized by hypotonic treatment or gently lysed with the detergent Brij-58. In the presence of KCl, EGTA, creatine phosphokinase, creatine phosphate, sucrose, dithiothreitol, CTP, GTP, UTP, and HEPES at pH 7.8, both in vitro systems required similar concentrations of all four deoxyribonucleoside triphosphates, ATP, Mg2+, and dextran. Incorporation of [3H] dTTP was due to semiconservative DNA replication and was restricted to S-phase nuclei. No repair replication was detectable. In the first 20 min, the rate of DNA replication in vitro was 30--40% of the in vivo rate, and after 60 min, about 3% of the genome were replicated. Preexisting bulk DNA was not fragmented during permeabilization or lysis with Brij-58 and during replication in vitro. Size distributions of growing strands in vitro were similar to those found in vivo. Neither cycloheximide (100 mug/ml) nor hydroxyurea (3 mM) inhibited DNA replication in vitro.

Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells.

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.