title: Isolation of human Dna2 endonuclease and characterization of its enzymatic properties.
abstract: In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase delta-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5' and 3' tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5' and 3' regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.
authors.affiliation: National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
authors.forename: Jeong-Hoon
authors.initials: JH
authors.lastname: Kim
authors.forename: Hee-Dai
authors.initials: HD
authors.lastname: Kim
authors.forename: Gi-Hyuck
authors.initials: GH
authors.lastname: Ryu
authors.forename: Do-Hyung
authors.initials: DH
authors.lastname: Kim
authors.forename: Jerard
authors.initials: J
authors.lastname: Hurwitz
authors.forename: Yeon-Soo
authors.initials: YS
authors.lastname: Seo
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; C022262:Okazaki fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C509908:DNA2 protein, human