Person:

Zhu, Bin

Gene 6 protein of bacteriophage T7 has 5′-3′-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5′-flap. This dependency of activity on the length of the 5′-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3′-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5′-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition.

An ideal DNA polymerase for chain-terminating DNA sequencing should possess the following features: (1) incorporate dideoxy- and other modified nucleotides at an efficiency similar to that of the cognate deoxynucleotides; (2) high processivity; (3) high fidelity in the absence of proofreading/exonuclease activity; and (4) production of clear and uniform signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally endowed with or can be engineered to have all these characteristics. The chemically or genetically modified enzyme (Sequenase) expedited significantly the development of DNA sequencing technology. This article reviews the history of studies on T7 DNA polymerase with emphasis on the serial key steps leading to its use in DNA sequencing. Lessons from the study and development of T7 DNA polymerase have and will continue to enlighten the characterization of novel DNA polymerases from newly discovered microbes and their modification for use in biotechnology.