title: DNA polymerase β contains a functional nuclear localization signal at its N-terminus.
abstract: DNA polymerase β (pol β) 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 N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), 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 β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β 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.
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Kirby
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Gassman
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Cassandra E
authors.initials: CE
authors.lastname: Smith
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Ming-Lang
authors.initials: ML
authors.lastname: Zhao
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.affiliation: National Institute of Environmental Health Sciences, Genome Integrity and Structural Biology Laboratory, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009154:Mutation; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D028901:alpha Karyopherins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D019951:DNA Polymerase beta