The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication

Nucleic Acids Res. 2015 Sep 18;43(16):7888-97. doi: 10.1093/nar/gkv710. Epub 2015 Jul 14.

Abstract

During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyltransferases / metabolism
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Cleavage
  • DNA Helicases / metabolism*
  • DNA Replication*
  • Deoxyribonucleases / metabolism*
  • Membrane Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Membrane Proteins
  • Okazaki fragments
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Acetyltransferases
  • ELO2 protein, S cerevisiae
  • Deoxyribonucleases
  • DNA Helicases
  • DNA2 protein, S cerevisiae