Isolation of human Dna2 endonuclease and characterization of its enzymatic properties.
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.
Kim
Jeong-Hoon
JH
National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
Kim
Hee-Dai
HD
Ryu
Gi-Hyuck
GH
Kim
Do-Hyung
DH
Hurwitz
Jerard
J
Seo
Yeon-Soo
YS
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
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