Metabolite Channelling in Deoxyribonucleotide and DNA Biosynthesis
Since my own research contributions lie primarily in this area, I will direct my commentary only to the first part of"Controversial Data"--section 6.2 of Dr. Ovfidi's article (Ov~idi, 1991). In this section she summarizes accurately, if incompletely, current research on enzyme organization in DNA precursor biosynthesis and its co-ordination with DNA replication. To be sure, the status of some work in this area remains unsettled, and in this commentary I will try to identify these unanswered questions, as well as topics upon which there seems to be general agreement.
It is useful to consider research in this area in terms of three distinct questions: (1) Are the enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis organized into multienzyme complexes? (2) Is the enzymatic machinery for dNTP synthesis juxtaposed with the DNA replication apparatus, so that the transfer of dNTPs to DNA polymerase active sites is kinetically facilitated? (3) Since the model of dNTP channeling has been developed largely in prokaryotic systems, how appropriate is the prokaryotic model to understanding DNA precursor dynamics in eukaryotic cells? I think that one can answer the first question in the affirmative. In T4 bacteriophage-infected Escherichia coil both our laboratory (cf. Mathews et al., 1988) and Greenberg's have described the partial purification of multienzyme aggregates containing at least ten enzymes of dNTP biosynthesis. Although recovery is low during purification, the aggregate retains through fractionation the ability to kinetically facilitate multi-step reaction pathways in oivo; i.e. the aggregate can catalyze sequences as long as six reactions, with appreciable shortening of transient times and lowering of intermediate concentrations, when compared with behavior expected for equivalent mixtures of non-complexed enzymes. Moreover, genetic evidence buttresses the kinetic data; when one enzyme in the complex is altered genetically, the alteration affects in vitro both the kinetics of multi-step pathways not involving the altered enzyme and the physical integrity of the complex Thyl~n & Mathews, 1989).
At least in one system, therefore, the evidence for existence ofa "dNTP synthetase" enzyme complex is reasonably strong. However, the question of whether this complex participates in channeling deoxyribonucleotides to the DNA replication machinery remains unsettled. Most of the evidence supporting this concept is indirect. Dr Ov~idi correctly cited our work that supports the existence of dNTP concentration gradients at replication sites in T4 phage-infected E. coli . Such gradients must be maintained in the absence of membranes to compartmentalize substrates in the face of exceedingly high dNTP pool turnover during replication.