Human DNA polymerase α in binary complex with a DNA:DNA template-primer

Sci Rep. 2016 Apr 1:6:23784. doi: 10.1038/srep23784.

Abstract

The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer--with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.

Publication types

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

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • DNA Primers
  • DNA Replication
  • Fluorescence Polarization
  • Humans
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Templates, Genetic

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • DNA
  • DNA Polymerase I