title: Flap endonuclease activity of gene 6 exonuclease of bacteriophage T7.
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.
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Mitsunobu
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.forename: Seung-Joo
authors.initials: SJ
authors.lastname: Lee
authors.forename: Stanley
authors.initials: S
authors.lastname: Tabor
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C022262:Okazaki fragments; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; D004247:DNA; D045585:Flap Endonucleases
keywords: Bacteriophage; Bacteriophage T7; DNA Damage; DNA Repair; DNA Replication; DNA-Protein Interaction; Flap Endonuclease; Gene 6 Exonuclease; Okazaki Fragment; RNA Processing