The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome

J Biol Chem. 2017 Jun 9;292(23):9627-9636. doi: 10.1074/jbc.M116.758524. Epub 2017 Apr 5.

Abstract

DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (Cdc45-MCM2-7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.

Keywords: AND-1; DNA polymerase; DNA replication; DNA-protein interaction; HMG domain; crystal structure; polymerase a; protein-protein interaction; replisome.

MeSH terms

  • DNA / biosynthesis
  • DNA / chemistry*
  • DNA / genetics
  • DNA Helicases / chemistry*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism
  • DNA Polymerase I / chemistry*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • DNA Replication / physiology
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism
  • Protein Domains
  • Protein Multimerization*

Substances

  • DNA-Binding Proteins
  • Multienzyme Complexes
  • WDHD1 protein, human
  • DNA
  • DNA Polymerase I
  • DNA Helicases

Associated data

  • PDB/4C8S
  • PDB/4C93
  • PDB/2D7L
  • PDB/5GVA
  • PDB/5GVB