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PMID:16595799

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Citation

Kim, JH, Kim, HD, Ryu, GH, Kim, DH, Hurwitz, J and Seo, YS (2006) Isolation of human Dna2 endonuclease and characterization of its enzymatic properties. Nucleic Acids Res. 34:1854-64

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.

Links

PubMed PMC1428795 Online version:10.1093/nar/gkl102

Keywords

Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/isolation & purification; Adenosine Triphosphatases/metabolism; Cell Line; DNA/metabolism; DNA Helicases/genetics; DNA Helicases/isolation & purification; DNA Helicases/metabolism; DNA, Single-Stranded/chemistry; DNA, Single-Stranded/metabolism; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/isolation & purification; Endodeoxyribonucleases/metabolism; Flap Endonucleases/metabolism; Humans; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:DNA2

enables

GO:0017116: single-stranded DNA helicase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:DNA2

NOT|enables

GO:0003678: DNA helicase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:DNA2

enables

GO:0017108: 5'-flap endonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:DNA2

enables

GO:0043142: single-stranded DNA-dependent ATPase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

Notes

See also

References

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