Sap1 Is a Replication-Initiation Factor Essential for the Assembly of Pre-Replicative Complex in the Fission Yeast Schizosaccharomyces pombe

J Biol Chem. 2017 Apr 14;292(15):6056-6075. doi: 10.1074/jbc.M116.767806. Epub 2017 Feb 21.

Abstract

A central step in the initiation of chromosomal DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) at late M and early G1 phase of the cell cycles. Since 1973, four proteins or protein complexes, including cell division control protein 6 (Cdc6)/Cdc18, minichromosome maintenance protein complex, origin recognition complex (ORC), and Cdt1, are known components of the pre-RC. Previously, we reported that a non-ORC protein binds to the essential element Δ9 of the Schizosaccharomyces pombe DNA-replication origin ARS3001. In this study, we identified that the non-ORC protein is Sap1. Like ORC, Sap1 binds to DNA origins during cell growth cycles. But unlike ORC, which binds to asymmetric AT-rich sequences through its nine AT-hook motifs, Sap1 preferentially binds to a DNA sequence of 5'-(A/T) n (C/G)(A/T)9-10(G/C)(A/T) n -3' (n ≥ 1). We also found that Sap1 and ORC physically interact. We further demonstrated that Sap1 is required for the assembly of the pre-RC because of its essential role in recruiting Cdc18 to DNA origins. Thus, we conclude that Sap1 is a replication-initiation factor that directly participates in the assembly of the pre-RC. DNA-replication origins in fission yeast are defined by possessing two essential elements with one bound by ORC and the other by Sap1.

Keywords: Cdc18; DNA; DNA binding protein; DNA origin; DNA replication; DNA replication initiation; DNA synthesis; DNA-protein interaction; Sap1; pre-RC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • DNA Replication / physiology*
  • DNA, Fungal / biosynthesis*
  • DNA, Fungal / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Nucleotide Motifs / physiology*
  • Replication Origin / physiology*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • Sap1 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • cdc18 protein, S pombe
  • cdc6 protein, S pombe