The N-terminal 45-kDa domain of Dna2 endonuclease/helicase targets the enzyme to secondary structure DNA.
The removal of initiating primers from the 5'-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea.
Chul-Hwan
CH
Lee
Miju
M
Lee
Hyo-Jin
HJ
Kang
Do-Hyung
DH
Kim
Young-Hoon
YH
Kang
Sung-Ho
SH
Bae
Yeon-Soo
YS
Seo
D004247:DNA / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000737:chemistry; D005819:Genetic Markers; D016678:Genome; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D005819:Genetic Markers; C022262:Okazaki fragments; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D003851:Deoxyribonucleases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae