dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency.
Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.
Barabás
Orsolya
O
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary.
Rumlová
Michaela
M
Erdei
Anna
A
Pongrácz
Veronika
V
Pichová
Iva
I
Vértessy
Beáta G
BG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004260:DNA Repair; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D008024:Ligands; D008027:Light; D016093:Mason-Pfizer monkey virus / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000378:metabolism*; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012542:Scattering, Radiation; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014961:X-Ray Diffraction; D014965:X-Rays
D008024:Ligands; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase