Interaction of the vaccinia virus nucleoside triphosphate phosphohydrolase I with linear oligonucleotides.
Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998) Genes Dev. 12, 538-546; Christen, L. M., et al. (1998) Virology 245, 360-371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure-function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparent Km for ATP is independent of DNA concentration, demonstrating that ssDNA binding alters the kcat for the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, the Kact decreases and the Vmax increases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.
Christen
L A
LA
Department of Biochemistry, Center for Microbial Pathogenesis, State University of New York, Buffalo 14214-3000, USA.
Sanders
M
M
Niles
E G
EG
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000998:Antiviral Agents / Q000235:genetics / Q000502:physiology; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000502:physiology; D004279:DNA, Viral / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D017874:Immediate-Early Proteins / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D043583:Nucleoside-Triphosphatase; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D013728:Terminator Regions, Genetic / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D000998:Antiviral Agents; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D017874:Immediate-Early Proteins; D009841:Oligonucleotides; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase