Variations in nuclear localization strategies among pol X family enzymes.
Variations in nuclear localization strategies among pol X family enzymes.
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DOI：
10.1111/tra.12600
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发表时间：
2018-06-22
期刊：
影响因子：
--
通讯作者：
London RE
中科院分区：
文献类型：
--
作者：
Kirby TW;Pedersen LC;Gabel SA;Gassman NR;London RE
尽管pol X家族酶在DNA修复中起着重要作用，但有关其核输入的结构基础的信息是有限的。最近的研究揭示了DNA聚合酶β中功能性NLS的意外存在，表明了主动核靶向的重要性，即使对于可能泄漏到细胞核内外的酶也是如此。目前的研究通过鉴定和结构表征三种剩余的人pol X酶中的功能性NLS序列来进一步探索这些酶的主动核转运：末端脱氧核苷酸转移酶（TdT）、DNA聚合酶μ（pol μ）和DNA聚合酶λ（pol λ）。NLS鉴定基于荧光素标记的NLS肽的荧光偏振测定的Importin α（Impα）结合亲和力、ImpαΔ伊布·NLS复合物的X射线晶体学分析和基于荧光的亚细胞定位研究。所有三种聚合酶都使用位于其N末端附近的NLS序列; TdT和pol μ利用单组分NLS序列，而pol λ利用在pol X家族成员中独特的双组分序列。pol μ NLS对Impα具有相对较弱的测定亲和力，部分原因是其靠近N-末端，限制了NLS之前侧翼残基的非特异性相互作用。然而，这种影响被部分减轻的N-末端序列不支持蛋氨酸氨肽酶的Met 1去除，导致3倍的亲和力增加时，N-末端蛋氨酸存在。核靶向对于每种pol X家族酶是独特的，其变化取决于每种聚合酶的结构和独特的功能作用。哺乳动物DNA聚合酶X在维持基因组完整性方面发挥重要作用，然而参与DNA修复需要核定位。我们在此报告：末端脱氧核苷酸转移酶、DNA聚合酶μ和DNA聚合酶λ（pol λ）核输入的结构基础;与Impα以及单个主要和次要Impα结合口袋结合的表观Kd值;以及验证核定位信号身份的功能研究。
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 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 characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase μ (pol μ), and DNA polymerase λ (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the ImpαΔIBB•NLS complexes, and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, 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. The mammalian DNA polymerase X enzymes play important roles in the maintenance of genome integrity, however participation in DNA repair requires nuclear localization. Here we report: the structural basis for nuclear import of terminal deoxynucleotidyl transferase, DNA polymerase μ, and DNA polymerase λ (pol λ); apparent Kd values for binding to Impα as well as to the individual major and minor Impα binding pockets; and functional studies verifying the identities of the nuclear localization signals.
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DOI：
10.1074/jbc.m110.102343
发表时间：
2010-06-04
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Giesecke A;Stewart M
通讯作者：
Stewart M
影响因子：
3.7
作者：
Braithwaite EK;Kedar PS;Stumpo DJ;Bertocci B;Freedman JH;Samson LD;Wilson SH
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影响因子：
14.9
作者：
Bernhofer M;Goldberg T;Wolf S;Ahmed M;Zaugg J;Boden M;Rost B
通讯作者：
Rost B
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
Cowtan, K
影响因子：
4.8
作者：
Ge, Qiang;Nakagawa, Tsutomu;Uyeda, Kosaku
通讯作者：
Uyeda, Kosaku