Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing.
Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.
Bae
Sung-Ho
SH
Department of Pharmacology, Dong-A University Cllege of Medicine, Seo-Gu, Busan, Korea.
Kim
Dong Wook
DW
Kim
Jiyoung
J
Kim
Jeong-Hoon
JH
Kim
Do-Hyung
DH
Kim
Hee-Dai
HD
Kang
Ho-Young
HY
Seo
Yeon-Soo
YS
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
C022262:Okazaki fragments; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D003850:Deoxyribonuclease I; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D008274:Magnesium