Abstract
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.
Highlights
Several specialized DNA polymerases are members of a novel polymerase family, the Y-family [3]
The UBZs Are Required for Enhanced Association between Pol and Monoubiquitinated proliferating cell nuclear antigen (PCNA)—Recent studies have demonstrated that monoubiquitination of PCNA in cells exposed to UV radiation promotes a more robust interaction of this accessory replication protein with Pol, Pol, and REV1 protein [10, 11, 25, 30]
Recent observations indicate that PCNA provides the central scaffold to which various Translesion DNA synthesis (TLS) polymerases can bind to access the replicative ensemble stalled at a lesion and to execute their roles in lesion bypass [32, 33]
Summary
Several specialized DNA polymerases are members of a novel polymerase family, the Y-family [3]. 3 The abbreviations used are: TLS, translesion DNA synthesis; UBZ, ubiquitinbinding zinc finger motif; Ub, ubiquitin; CHX, cycloheximide; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen; BPDE, benzo[a]pyrene-dihydrodiol epoxide; mPol, mouse Pol; hPol, human Pol; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; MEF, mouse embryonic fibroblast. Protein Half-life Determination—COS7 cells were transfected with the wild-type and UBZs mutant HA-Pol constructs.
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