Requirements for the interaction of mouse Polkappa with ubiquitin and its biological significance.
Polkappa protein is a eukaryotic member of the DinB/Polkappa branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polkappa in this process has remained unknown. Here we report that mouse Polkappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polkappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polkappa. In addition, Polkappa binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polkappa. The ubiquitin-binding motifs are also required for Polkappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polkappa half-life. Finally, we have examined levels of Polkappa expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation.
Guo
Caixia
C
Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9072, USA.
Tang
Tie-Shan
TS
Bienko
Marzena
M
Dikic
Ivan
I
Friedberg
Errol C
EC
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; C498613:Polk protein, mouse; D000099270:Y-Family DNA Polymerases