Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

Nucleic Acids Res. 2017 Sep 6;45(15):9046-9058. doi: 10.1093/nar/gkx633.

Abstract

We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.

MeSH terms

  • Amino Acid Substitution*
  • Arginine / chemistry
  • Arginine / metabolism
  • Biological Assay*
  • Cloning, Molecular
  • DNA Primase / genetics*
  • DNA Primase / metabolism
  • DNA Primers / chemical synthesis
  • DNA Primers / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fluorescent Dyes / chemistry
  • Fluorometry / methods
  • Gene Expression
  • Gene Library
  • Humans
  • Multifunctional Enzymes / genetics*
  • Multifunctional Enzymes / metabolism
  • Mutation
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism
  • Organic Chemicals / chemistry
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Multimerization
  • RNA / genetics*
  • RNA / metabolism
  • RNA-Directed DNA Polymerase / genetics*
  • RNA-Directed DNA Polymerase / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Tyrosine / chemistry
  • Tyrosine / metabolism

Substances

  • DNA Primers
  • Fluorescent Dyes
  • Multifunctional Enzymes
  • Oligonucleotides
  • Organic Chemicals
  • SYBR Green I
  • Tyrosine
  • RNA
  • Arginine
  • DNA Primase
  • PrimPol protein, human
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase