Portal de Periódicos da CAPES

T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups.

1983; Elsevier BV; Volume: 258; Issue: 11 Linguagem: Inglês

10.1016/s0021-9258(18)32317-2

1083-351X

S. P. Adler, Paul Modrich,

Carcinogens and Genotoxicity Assessment

Bacteriophage T7-induced DNA polymerase is composed of a 1: 1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin.Preparation of active subunits in the absence of sulfbydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme.The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by Nethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein.Thioredoxin sulfhydryl groups present in native T7 DNA polymerase do not appear to be involved in an intersubunit disulfide bond; one and probably both sulfhydryls are available in the native holoenzyme for modification by N-ethylmaleimide.Furthermore, DNA substrates alter the reactivity of thioredoxin cysteines within the holoenzyme with respect to this reagent.Substrates for the single strand exonuclease enhance the reactivity of thioredoxin sulfhydryl groups while those for the polymerase or double strand exonuclease functions afford protection.It, therefore, seems likely that thioredoxin sulfhydryl groups are present in the reduced state within the native polymerase.The DNA polymerase induced by bacteriophage T7 is a 1:l complex of two subunits, an Mr = 80,000 protein encoded by viral gene 5 and M, = 12,000 Escherichia coli thioredoxin, both of which are required for viral DNA synthesis in vivo (1)(2)(3)(4)(5).Previous studies have demonstrated that the native holoenzyme possesses three catalytic activities: DNA polymerase, a 3'-to 5'-exonuclease which degrades single-stranded DNA, and a 3'-to 5'-exonuclease which hydrolyzes duplex DNA.While the isolated viral polypeptide has been found to possess normal levels of the 3' to 5' single strand exonuclease, DNA polymerase and 3' to 5' double strand hydrolytic activities are dependent on the presence of both phage and host specified subunits (3, 4, 6, 7).In addition to its role in T7 DNA biosynthesis, in vitro studies have implicated thioredoxin in a variety of metabolic processes.The E. coli protein was initially identified as an oxidation-reduction cofactor for ribonucleoside diphosphate