The role of DNA polymerase ι in UV mutational spectra
The role of DNA polymerase ι in UV mutational spectra
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DOI：
10.1016/j.mrfmmm.2006.01.003
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发表时间：
2006-07-25
影响因子：
2.3
通讯作者：
Pfeifer, Gerd P.
中科院分区：
文献类型：
--
作者：
Choi, Jun-Hyuk;Besaratinia, Ahmad;Pfeifer, Gerd P.
UVB(280-320 nm)和UVC(200-280 nm)辐射在DNA中主要产生环丁烷嘧啶二聚体(CPDS)和(6-4)光产物。CPDS被认为是紫外线诱导的大部分突变的原因。胸腺嘧啶-胸腺嘧啶CPD，以及可能也含有胞嘧啶的CPD，通过依赖DNA聚合酶ETA(PolETA)的过程在体内以基本准确的方式复制。POL ETA是一种DNA损伤耐受和易出错的DNA聚合酶，由人类的POLH(XPV)基因编码。容易出错的DNA聚合酶Y家族的另一个成员是编码DNA聚合酶IOTA的PolI(Poli)。为了阐明Poliota在紫外线诱变中的特定作用，我们使用了siRNA敲除方法与在哺乳动物细胞中复制的supF穿梭载体相结合，类似于我们之前对PoleTA所做的操作。合成的RNA双链被用来有效地抑制293T细胞中Pol iota的表达。用254nmUVC照射supF穿梭载体，并在293T细胞中复制。令人惊讶的是，从带有Pol iota基因敲除的细胞中回收的质粒持续减少，这与紫外线照射没有关系。在siRNA敲除细胞中，supF突变频率与对照细胞相比没有变化，证实了Poliota在紫外线诱变中不起重要作用。从siRNA处理的细胞和对照细胞中测定了紫外线诱导的supF突变体的序列。在对照细胞和siRNA基因敲除细胞中，supF基因的突变类型和分布均无显著差异，主要是双嘧啶的C到T和CC到TT的转变。这些结果表明，Poliota在体内的紫外线损伤旁路和诱变中没有明显的作用，并提供了一些初步数据，表明该聚合酶可能参与染色体外DNA的复制。(C)2006爱思唯尔B.V.保留所有权利。
UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol iota). In order to clarify the specific role of Pol iota in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol eta. Synthetic RNA duplexes were used to efficiently inhibit Pol iota expression in 293T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293T cells in presence of anti-Pol iota siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol iota knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol iota does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol iota has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA. (c) 2006 Elsevier B.V. All rights reserved.