Isolation of human Dna2 endonuclease and characterization of its enzymatic properties

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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.
Publisher
Oxford Univ Press
Issue Date
2006-04
Language
English
Article Type
Article
Keywords

OKAZAKI FRAGMENT MATURATION; CELL NUCLEAR ANTIGEN; SACCHAROMYCES-CEREVISIAE DNA2; REPLICATION PROTEIN-A; SINGLE-STRANDED-DNA; ESSENTIAL IN-VIVO; LAGGING-STRAND; CAENORHABDITIS-ELEGANS; FLAP ENDONUCLEASE-1; POLYMERASE-DELTA

Citation

NUCLEIC ACIDS RESEARCH , v.34, no.6, pp.1854 - 1864

ISSN
0305-1048
DOI
10.1093/nar/gkl102
URI
http://hdl.handle.net/10203/90447
Appears in Collection
BS-Journal Papers(저널논문)
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