The characterization of a mammalian DNA structure-specific endonuclease.
The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.
Harrington
J J
JJ
Department of Pathology, Stanford University School of Medicine, CA 94305-5324.
Lieber
M R
MR
D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair; D017886:Durapatite; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism*; D045585:Flap Endonucleases; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D017886:Durapatite; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases