Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis

Nucleic Acids Res. 1996 Dec 15;24(24):4845-52. doi: 10.1093/nar/24.24.4845.


The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopolymers
  • Cloning, Molecular
  • DNA Polymerase I / chemistry*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • Escherichia coli
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics
  • Mutagenesis
  • Mycobacterium tuberculosis / enzymology*
  • Oligodeoxyribonucleotides / chemistry*


  • Biopolymers
  • Oligodeoxyribonucleotides
  • DNA Polymerase I
  • Exodeoxyribonucleases