title: Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: 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
authors.forename: V
authors.initials: V
authors.lastname: Mizrahi
authors.forename: P
authors.initials: P
authors.lastname: Huberts
mesh_terms: D001704:Biopolymers; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
chemical_list: D001704:Biopolymers; D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases