A DNA polymerase-{alpha}{middle dot}primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells

J Biol Chem. 2009 Feb 27;284(9):5807-18. doi: 10.1074/jbc.M807593200. Epub 2008 Dec 31.


alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Northern
  • Cells, Cultured
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA Primase / genetics
  • DNA Primase / metabolism*
  • DNA Replication*
  • Fluorescent Antibody Technique
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Kidney / cytology
  • Kidney / metabolism
  • Leukemia L1210 / metabolism
  • Leukemia L1210 / pathology
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phylogeny
  • Protein Biosynthesis
  • Protein Multimerization
  • Proteins / antagonists & inhibitors
  • Proteins / genetics
  • Proteins / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / pharmacology
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Transcription, Genetic
  • Transfection


  • Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • Replication Protein A
  • DNA Primase
  • DNA polymerase alpha-primase
  • DNA Polymerase I