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Abstract
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.
Highlights
We recently reported that the primase domain of the 56-kDa T7 gene 4 protein can use preformed oligonucleotides and even tRNA as functional primers for T7 DNA polymerase (24)
T7 DNA polymerase deficient in the 3Ј–5Ј-exonuclease activity does catalyze rather extensive strand displacement synthesis leading to 5Ј-single-stranded termini when synthesis encounters a duplex structure (23)
On the basis of the above observations we have examined the properties of gp6 to determine if it has flap endonuclease activity in addition to its 5Ј–3Ј-exonuclease activity
Summary
Conclusion: The flap endonuclease activity catalyzes the removal 5Ј-single-stranded tails arising from duplex DNA. Gp can hydrolyze a RNA strand in an RNA/DNA hybrid (9, 10), and purified gp catalyzes the removal of the tetraribonucleotide primers at the 5Ј-termini of fragments arising from the extension of RNA primers synthesized by gene 4 DNA primase (11) The products of this reaction are ATP and nucleoside 5Ј-monophosphates. T7 DNA polymerase deficient in the 3Ј–5Ј-exonuclease activity does catalyze rather extensive strand displacement synthesis leading to 5Ј-single-stranded termini when synthesis encounters a duplex structure (23). We show that gp does have a flap endonuclease activity that removes 5Ј-ssDNA termini on duplex DNA as well as ssDNA flaps of the type that arise from strand displacement synthesis. The finding that gp has flap endonuclease activity provides an explanation for the early observation that it can degrade phage DNA, DNA has an ssDNA tail, and gp does not normally hydrolyze ssDNA (1)
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