An exposed cysteine of T7 gene 5 are critical for interaction with its processive factor, thioredoxin Academic Article uri icon

abstract

  • DNA polymerase (gp5) of bacteriophage T7 is a non-processive polymerase encoded by gene 5 of the phage. Escherichia coli encoded thioredoxin (trx) binds tightly to the thumb subdomain of gp5 (gp5/trx) and increases processivity to approximately 800 nucleotides per binding event. Gene 5 protein has ten cysteine residues, of which only Cys275 and Cys313 are exposed on the protein surface; both residues are located on a flexible loop of the thioredoxin binding domain. In the present study, we replaced either Cys275 or Cys313 with a serine to examine the role of these two exposed cysteines on the function of T7 DNA polymerase. Replacement of Cys275 with a serine (gp5-C275S/trx) has little effect on DNA polymerase activity as compared with wild-type gp5/trx. However, substitution of a serine for Cys313 decreases DNA polymerase activity 3-to 4-fold on linear and circular primed DNA …

publication date

  • April 1, 2011