The function of thioredoxin and glutathione in deoxyribonucleic acid synthesis.

Ribonucleotide reductases (EC 1 .I 7.4.1 and EC 1.17.4.2) catalyse the reduction of ribonucleoside 5’-diphosphates or triphosphates to the corresponding 2’-deoxyribonucleotides and are key enzymes for the onset of DNA replication during the cell cycle (Reichard, 1968; Follmann, 1974). The reduction of the 2’-hydroxyl group of the ribose moiety in a ribonucleotide is dependent on NADPH, but this coenzyme will not function directly with ribonucleotide reductase. In vitro, purified ribonucleotide reductase requires certain dithiols like lipoic acid or dithiothreitol for activity (Reichard, 1968). The thioredoxin system of Escherichia coli was discovered in 1964 by Reichard and coworkers (Laurent et al., 1964) as a physiological NADPH-dependent hydrogen-donor systemforribonucleotidereductase. Thioredoxin is asmall acidic dithiol protein (mol.wt. 12000) which can occur in an oxidized (thioredoxin-S,) or a reduced [thioredoxin-(SH),] form. Thioredoxin reductase is a flavoprotein (mol.wt. 66000) which will reduce thioredoxin-S, by NADPH (Reichard, 1968). Ribonucleotide reduction as studied in vitro thus appears as a complex multienzyme system. Further, evidence exists that the organization in vivo of the ribonucleotide reductase system may involve interactions between the proteins to form a highly effective multienzyme complex of unknown properties. This is supported by the low activity of ribonucleotide reductase obtained by reconstitution in vitro as compared with the activity in cells made permeable to nucleotides by diethyl ether treatment or the activity in ‘high-speed’ supernatants from gently lysed E. coli cells (Eriksson, 1975). Steady-state kinetics of ribonucleotide reductase indicated that the enzyme acted by a Ping Pong mechanism, in the presence of thioredoxin-(SH)z, that is, it akernated between two stable forms during catalysis (Thelander, 1974). The electron acceptor in ribonucleotide reductase was suggested to be oxidation-reduction-active disulphides of protein B1 (Thelander, 1974). The Ping Pong mechanism might explain the difliculties in observing any binding between Escherichia coli thioredoxin and ribonucleotide reductase in vitro (A. Holmgren, unpublished work). From an X-ray-crystallographic study of E. coli thioredoxin-S, to 0.28 nm (2.8A) resolution, the oxidation-reductionactive disulphide bond has been located in a protrusion of the molecule (Holmgren et al., 1975). Reduction of thioredoxin-Sz is suggested to be accompanied by a local conformational change within this active-centre protrusion (Holmgren et al., 1975). From such a model the simultaneous binding of thioredoxin to thioredoxin reductase and ribonucleotide reductase seems unlikely. Both these enzymes have oxidationreduction disulphide bonds at their active sites (Thelander, 1974) that should interact with the S-S bond protrusion in thioredoxin for transfer of electrons via thiol-disulphide interchange. The structural data for thioredoxin thus also support Ping Pong mechanisms for the interaction of thioredoxin with both ribonucleotide and thioredoxin reductase. A completely new and unexpected role for thioredoxin in DNA synthesis was discovered in the work of Richardson and co-workers (Mark & Richardson, 1976). E. coli thioredoxin was found to be a subunit of a phage TFinduced DNA polymerase, detected after virus infection, which is essential for the phage life cycle. Phage T7 DNA polymerase is composed of one 84000-dalton phage-coded subunit and 1 molecule of thioredoxin (Mark & Richardson, 1976). The specific function of thioredoxin in the phage T7 DNA polymerase is presently unknown. However, since the enzyme can be reconstituted in vitro from purified gene-5 protein of phage T7 by addition of E. coli thioredoxin, the function of the thioredoxin subunit can be studied in some detail. The discovery of the new function for E. coli thioredoxin as subunit of phage T7 DNA polymerase was based on the isolation of E. coli mutants (tsnC) which could not