Spatial relationship between polymerase and exonuclease active sites of phage T4 DNA polymerase enzyme

The spatial relationship between the polymerase and exonuclease active sites of bacteriophage T4 DNA polymerase enzyme has been examined using a bulky biotin-streptavidin block at a specified position in an oligonucleotide (Fig. 1). The idea was to monitor the closest distance of approach of the T4 enzyme before being blocked by the bulky biotin-streptavidin complex while performing either of its activities. The results indicated a distance of 4-5 nucleotides between the biotin-streptavidin probe and the exonuclease site and a distance requirement of at least 7 nucleotides between the bulky probe and the 3'-primer terminus for efficient polymerization by the T4 enzyme. The difference in the two distances suggested a separation of 2-3 nucleotides between the two active sites of the T4 enzyme.