Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA.
E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.
Kumar
Pradeep
P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Krishna
Kurthkoti
K
Srinivasan
Ramanujam
R
Ajitkumar
Parthasarathi
P
Varshney
Umesh
U
D001483:Base Sequence; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014498:Uracil / Q000737:chemistry*; D051981:Uracil-DNA Glycosidase
D004269:DNA, Bacterial; D014498:Uracil; D009701:Nucleoside-Diphosphate Kinase; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase