Nucleic Acids Research, Vol 24, Issue 24 4845-4852, Copyright © 1996 by Oxford University Press
V Mizrahi and P Huberts
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.
ARTICLES
Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis
Molecular Biology Unit, South African Institute for Medical Research and Department of Hematology, University of the Witwatersrand Medical School, Johannesburg, South Africa. 075val@chiron.wits.ac.za
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