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Variations in nuclear localization strategies among pol X family enzymes.

ABSTRACT: Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase ?, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol ?) and DNA polymerase lambda (pol ?). NLS identifications are based on Importin ? (Imp?) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Imp??IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol ? utilize monopartite NLS sequences, while pol ? utilizes a bipartite sequence, unique among the pol X family members. The pol ? NLS has relatively weak measured affinity for Imp?, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

SUBMITTER: Kirby TW

PROVIDER: S-EPMC6684861 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Variations in nuclear localization strategies among pol X family enzymes.

Kirby Thomas W TW Pedersen Lars C LC Gabel Scott A SA Gassman Natalie R NR London Robert E RE

Traffic (Copenhagen, Denmark) 20180622

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally character ...[more]

PMID: 29931796

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Project description:BackgroundThe nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system.ResultsIn this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families.ConclusionThe results of this study reveal that Planctomycetaceae separates slightly from the members of non-planctomycete bacteria but still has substantial differences from fungi, based on the NLS-like motifs and NLS-bearing protein analysis.

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Variations in nuclear localization strategies among pol X family enzymes.

Publications

Variations in nuclear localization strategies among pol X family enzymes.

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