Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7
Author
Mitsunobu, Hitoshi
Zhu, Bin
Lee, Seung-Joo
Tabor, Stanley
Richardson, Charles C.
Published Version
https://doi.org/10.1074/jbc.M113.538611Metadata
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Mitsunobu, Hitoshi, Bin Zhu, Seung-Joo Lee, Stanley Tabor, and Charles C. Richardson. 2014. “Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7.” Journal of Biological Chemistry 289 (9): 5860–75. https://doi.org/10.1074/jbc.m113.538611.Abstract
Background: Flap endonucleases remove 5-single-stranded DNA termini. Results: T7 gene 6 exonuclease is a flap endonuclease that cleaves 5-single-stranded termini one nucleotide into the duplex. Conclusion: The flap endonuclease activity catalyzes the removal 5-single-stranded tails arising from duplex DNA. Significance: The specificity of gene 6 protein identifies it as a flap endonuclease that can remove unusual structures of recombination and replication.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 dsDNA-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 gene 6 protein 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 because 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.Terms of Use
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