PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching
PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching
复制标题
DOI：
10.1371/journal.pone.0213383
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发表时间：
2019-03
期刊：
影响因子：
3.7
通讯作者：
M. Tsuda;Saki Ogawa;Masato Ooka;Kaori Kobayashi;K. Hirota;M. Wakasugi;T. Matsunaga;Tetsushi Sakuma;Takashi Yamamoto;S. Chikuma;H. Sasanuma;M. Debatisse;A. Doherty;R. Fuchs;S. Takeda
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文献类型：
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作者：
M. Tsuda;Saki Ogawa;Masato Ooka;Kaori Kobayashi;K. Hirota;M. Wakasugi;T. Matsunaga;Tetsushi Sakuma;Takashi Yamamoto;S. Chikuma;H. Sasanuma;M. Debatisse;A. Doherty;R. Fuchs;S. Takeda
复制型DNA聚合酶经常在受损的模板链上停滞。停滞的复制叉通过DNA损伤耐受（DDT）途径、易错跨损伤DNA合成（TLS）来科普过度的DNA损伤以及通过同源DNA重组进行无错误模板切换（TS）来恢复。PDIP 38（38 kDa的Pol-delta相互作用蛋白），也称为Pol δ相互作用蛋白2（PolDIP 2），与TLS DNA聚合酶、聚合酶η（Polη）、Polλ和PrimPol物理缔合，并在体外激活它们。目前还不清楚PDIP 38是否在体内促进TLS，因为没有方法可以测量哺乳动物细胞中的单个TLS事件。我们破坏了PDIP 38基因，从鸡DT 40和人TK 6 B细胞系中产生了PDIP 38-/-细胞。这些PDIP 38-/-细胞没有显示出对UV或H2 O2的显著敏感性，这是在任何TLS聚合酶缺陷型DT 40或TK 6突变体中未观察到的表型。由于细胞在体外培养期间通过TLS（非模板化IG V超突变）和TS（IG基因转换）不断使IG V多样化，因此DT 40提供了通过免疫球蛋白可变（IG V）基因的核苷酸序列分析检查个体TLS和TS事件的独特机会。PDIP 38-/-细胞显示出IG V多样化从TLS到TS的转变。我们测量了TK 6细胞中TLS和TS在化学合成的UV损伤（CPD）整合到基因组DNA中的相对使用。PDIP 38的丢失也导致TS的相对使用增加。在PDIP 38-/-人和鸡细胞中，UV诱导的姐妹染色单体交换（与交叉相关的TS事件）的数量增加了几倍。总的来说，PDIP 38的丢失始终导致DDT从TLS转变为TS，而不会增强细胞对DNA损伤的敏感性。我们建议PDIP 38控制TLS和TS的相对使用，增加TLS的使用，而不改变DDT的整体能力。
Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.