Multienzyme complexes in DNA precursor biosynthesis

Abstract

Several features of DNA replication suggest the involvement of multienzyme complexes in deoxyribonucleotide biosynthesis. Such complexes could generate intracellular concentration gradients of DNA precursors, with high levels being maintained at their sites of utilization, namely, replication forks. Relevant facts or observations include the following. (1) Deoxyribonucleotides are highly specialized metabolites, whose sole evident function is to serve as DNA precursors; for the most part they are found only in cells which are synthesizing DNA. (2) Although average intracellular deoxyribonucleoside triphosphate concentrations are low, most in vitro systems for DNA synthesis require rather high triphosphate concentrations to maintain maximal incorporation rates; thus, the effective intracellular levels are probably much higher than the average concentrations; (3) DNA chain growth rates in vivo are one to two orders of magnitude higher than the corresponding rates for RNA chain growth; thus, DNA precursor supplies must be replenished very rapidly at the replication fork. Data on deoxyribonucleotide pool labeling kinetics indicate that DNA precursor pools are compartmentalized. Even in the absence of physically separated compartments, one can visualize a functional compartmentation in which the separate pools are kinetically distinct. In bacteria, for example, one could envisage a small, rapidly turning over pool of triphosphates near the replication forks, and a larger, more slowly replenished pool which is more evenly distributed through the cell and which is used primarily in DNA repair. A complex of deoxyribonucleotide-synthesizing enzymes, juxtaposed with the replication apparatus, could serve to maintain high local concentrations of triphosphates at the replication forks. Using T4 phage-infected E. coli as a model system, our laboratory has obtained considerable evidence supporting the above model. The evidence is derived from deoxyribonucleotide pool kinetic studies, experiments on DNA synthesis in vitro, and properties of an isolated multienzyme aggregate. The evidence indicates that effective DNA precursor concentrations at the replication fork are quite different from average intracellular concentrations as estimated from pool measurements. The desirability of being able to determine effective concentrations is emphasized.