Characterization of human cytomegalovirus uracil DNA glycosylase (UL114) and its interaction with polymerase processivity factor (UL44).
Here, we report the molecular characterization of the human cytomegalovirus uracil DNA glycosylase (UNG) UL114. Purified UL114 was shown to be a DNA glycosylase, which removes uracil from double-stranded and single-stranded DNA. However, kinetic analysis has shown that viral UNG removed uracil more slowly compared with the core form of human UNG (Delta84hUNG), which has a catalytic efficiency (k(cat)/K(M)) 350- to 650-fold higher than that of UL114. Furthermore, UL114 showed a maximum level of DNA glycosylase activity at equimolar concentrations of the viral polymerase processivity factor UL44. Next, UL114 was coprecipitated with DNA immobilized to magnetic beads only in the presence of UL44, suggesting that UL44 facilitated the loading of UL114 on DNA. Moreover, mutant analysis demonstrated that the C-terminal part of UL44 (residues 291-433) is important for the interplay with UL114. Immunofluorescence microscopy revealed that UL44 and UL114 colocalized in numerous small punctuate foci at the immediate-early (5 and 8 hpi) phases of infection and that these foci grew in size throughout the infection. Furthermore, coimmunoprecipitation assays with cellular extracts of infected cells confirmed that UL44 associated with UL114. Finally, the nuclear concentration of UL114 was estimated to be 5- to 10-fold higher than that of UL44 in infected cells, which indicated a UL44-independent role of UL114. In summary, our data have demonstrated a catalytically inefficient viral UNG that was highly enriched in viral replication foci, thus supporting an important role of UL114 in replication rather than repair of the viral genome.
Ranneberg-Nilsen
Toril
T
Department of Virology, Rikshospitalet Medical Centre, N-0027 Oslo, Norway.
Dale
Hege Avsnes
HA
Luna
Luisa
L
Slettebakk
Ragnhild
R
Sundheim
Ottar
O
Rollag
Halvor
H
Bjørås
Magnar
M
D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D011485:Protein Binding; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D014764:Viral Proteins; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase