DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.
DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.
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DOI：
10.1371/journal.pone.0012229
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发表时间：
2010-08-18
期刊：
影响因子：
3.7
通讯作者：
Wilson SH
中科院分区：
文献类型：
--
作者：
Braithwaite EK;Kedar PS;Stumpo DJ;Bertocci B;Freedman JH;Samson LD;Wilson SH
碱基切除修复（BER）是一种DNA修复途径，旨在纠正基因组DNA中的小碱基损伤。虽然已知DNA聚合酶β（pol β）是BER途径中的主要聚合酶，但各种研究表明其他DNA聚合酶起着后备作用。一种这样的聚合酶，DNA聚合酶λ（pol λ），被证明在氧化DNA损伤的BER中是重要的。为了进一步探索X家族DNA聚合酶λ和β在BER中的作用，我们制备了一种pol β和pol λ基因缺失的小鼠胚胎成纤维细胞系。中性红活力测定表明pol λ和pol β双缺失细胞对烷化剂和氧化性DNA损伤剂高度敏感。体外BER测定揭示了pol λ对碱基损伤的单核苷酸BER的适度贡献。此外，使用纯化的酶和全细胞提取物的免疫共沉淀实验，我们发现pol λ和pol β都与上游DNA糖基化酶相互作用，用于修复烷基化和氧化的DNA碱基。这种相互作用可能是重要的协调作用，这些聚合酶在BER。
Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.
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