Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7 [DNA and Chromosomes]

January 6th, 2014 by Mitsunobu, H., Zhu, B., Lee, S.-J., Tabor, S., Richardson, C. C.

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′-3′ exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a ds/ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gp6 flap endonuclease activity. The released flap is not degraded further but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins since Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.