DNA polymerase beta contains a functional nuclear localization signal at its N-terminus

摘要

DNA polymerase beta (pol beta) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the Nterminal lyase domain. Binding of this domain to Importin alpha 1 (Imp alpha 1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol beta residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar K-d, that is selective for the murine Importin alpha 1 (mImp alpha 1) minor site, with the Kd strengthening to similar to 140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin alpha 5 (hImp alpha 5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol beta (R4S, K5S) in mouse embryonic fibroblasts lacking endogenous pol beta. Together these data demonstrate that pol beta contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.