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A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.

Pubmed ID: 22593576

Authors

  • Kilkenny ML
  • De Piccoli G
  • Perera RL
  • Labib K
  • Pellegrini L

Journal

The Journal of biological chemistry

Publication Data

July 6, 2012

Associated Grants

  • Agency: Wellcome Trust, Id: 084279
  • Agency: Wellcome Trust, Id:

Mesh Terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Conserved Sequence
  • DNA Polymerase I
  • DNA Primase
  • DNA Replication
  • Eukaryotic Cells
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Peptides
  • Protein Binding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Sequence Homology, Amino Acid