Characterization of the DNA damage-inducible helicase DinG from Escherichia coli.
The dinG promoter was first isolated in a genetic screen scoring for damage-inducible loci in Escherichia coli (Lewis, L. K., Jenkins, M. E., and Mount, D. W. (1992) J. Bacteriol. 174, 3377-3385). Sequence analysis suggests that the dinG gene encodes a putative helicase related to a group of eukaryotic helicases that includes mammalian XPD (Koonin, E. V. (1993) Nucleic Acids Res. 21, 1497), an enzyme involved in transcription-coupled nucleotide excision repair and basal transcription. We have characterized the dinG gene product from E. coli using genetic and biochemical approaches. Deletion of dinG has no severe phenotype, indicating that it is non-essential for cell viability. Both dinG deletion and over-expression of the DinG protein from a multicopy plasmid result in a slight reduction of UV resistance. DinG, purified as a fusion protein from E. coli cells, behaves as a monomer in solution, as judged from gel filtration experiments. DinG is an ATP-hydrolyzing enzyme; single-stranded (ss) DNA stimulates the ATPase activity 15-fold. Kinetic data yield a Hill coefficient of 1, consistent with one ATP-hydrolyzing site per DinG molecule. DinG possesses a DNA helicase activity; it translocates along ssDNA in a 5' --> 3' direction, as revealed in experiments with substrates containing non-natural 5'-5' and 3'-3' linkages. The ATP-dependent DNA helicase activity of DinG requires divalent cations (Mg2+, Ca2+, and Mn2+) but is not observed in the presence of Zn2+. The DinG helicase does not discriminate between ribonucleotide and deoxyribonucleotide triphosphates, and it unwinds duplex DNA with similar efficiency in the presence of ATP or dATP. We discuss the possible involvement of the DinG helicase in DNA replication and repair processes.
Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Oleg N
ON
Voloshin
Filip
F
Vanevski
Pavel P
PP
Khil
R Daniel
RD
Camerini-Otero
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations; D002470:Cell Survival; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002412:Cations; D029968:Escherichia coli Proteins; C074432:dinG protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA