Thioredoxins from spinach chloroplasts supplement phage T7 gene 5 protein for DNA polymerase activity

Thioredoxins are ubiquitous low Mr hydrogen carrier proteins. This class of proteins has first been described as a hydrogen donor for the ribonucleotide reductase in Escherichia coli [ 11. It has since been shown that thioredoxins have various functions in the cellular metabolism [2]. Their function suggests also evolutionary conservation, but they differ in size and in amino acid sequence, when isolated from various sources. In the chloroplasts of green plants thioredoxins are part of a light-dependent activation mechanism for some key enzymes [3]. In the light, electrons from chlorophyll reduce ferredoxin and the reduced ferredoxin transfers its electrons via the enzyme ferredoxin-thioredoxin reductase to thioredoxin which then activates certain enzymes like fructose 1 $bisphosphatase and NADP-dependent malate dehydrogenase. The thioredoxins from spinach chloroplasts have been purified and resolved into 3 different proteins: thioredoxin j-(1 1 100 Mr), capable of activating chloroplast fructose 1 $bisphosphatase and malate dehydrogenase, thioredoxin mc (10 7OOMr) and thioredoxin mb (10 SOOM,), both capable of activating only malate dehydrogenase. These thioredoxins have been characterized by amino acid compositions and N-terminal and C-terminal sequences. The results allowed us to conclude that thioredoxin fis a protein different from the m-type thioredoxins, whereas thioredoxin mb and mc are N-terminal redundant isomers [4]. Thioredoxin-Sz (11 700 Mr) from E. coli is one of the best characterized thioredoxins [S]. The complete sequence and the 2.8 A three-dimensional structure are known and show the presence of the catalytically active cystine S-S bridge. The size and amino acid composition differs from the 3 spinach thioredoxins. The DNA polymerase induced by phage T7 consists of 2 subunits, an 84 000 I& protein encoded by the phage gene 5 and a 12 000 MI protein, which is the thioredoxin of the E. coli cell [6]. This protein complex could be separated into its subunits and subsequently reconstituted [7]. Phage T7 gene 5 protein could be supplemented in vitro with E. coli thioredoxin to yield fully active T7 DNA polymerase [8]. The purified gene 5 protein still exhibits enzymatic activities as the 3’-5’ hydrolysis of single-stranded DNA [9]. Hydrolysis of duplex DNA, however, requires as the DNA polymerase activity the presence of thioredoxin. For replication of duplex DNA, T7 DNA polymerase interacts with the phage-encoded gene 4 protein [lo]. This enzyme system hydrolyses nucleoside triphosphates and unwinds nicked duplex DNA. It has been used to replicate specifically cleaved phage fd RF [ll]. It was of interest to see whether thioredoxins from spinach chloroplasts whose prime function is assumed to be the activation of photosynthetic enzymes could substitute for the E. coli thioredoxin in the T7 DNA polymerase and whether this system distinguishes between the 3 thioredoxins.