Molecular mechanisms of mutagenesis caused by transletion DNA synthesis

DNA复制装置在遇到模板上的DNA损伤时停止复制。细胞识别出这样的复制分叉块，并开始操作一些细胞功能来克服复制分叉块。在大肠杆菌细胞中，DNA复制叉阻断可诱导复制后重组修复和SOS修复。最近，这些功能似乎也在真核细胞中起作用。本项目旨在阐明大肠杆菌和高等真核生物翻译DNA合成的分子机制。利用大肠杆菌复制装置进行体外DNA复制的诱变试验：我们建立了几种由纯化的复制酶重建的体外DNA复制系统，并成功地测定了DNA复制引起的正向突变。碱基取代突变似乎是由自发DNA损伤引起的DNA复制错误在多个热点上引起的。此外，发现复制装置最常见的复制错误是那些导致单碱基帧移的错误。这种类型的复制错误是在模板序列中相同核苷酸的运行中高度特异性地产生的。利用体外DNA复制系统分析天然DNA链延伸暂停位点：以M13单链噬菌体DNA为模板，利用大肠杆菌DNA聚合酶III全酶研究体外系统中DNA链延伸暂停位点。共发现8个强停顿区，每个停顿区由多个停顿点组成。大约一半的暂停位点能够形成稳定的发夹结构。鉴定非洲爪蟾卵母细胞提取物中涉及翻译DNA合成的因素：利用合成的含有特定位点DNA损伤的低聚物DNA，我们搜索了翻译DNA合成的酶活性。至少发现了三种不同的途径。

The DNA replication apparatus stops when it meets DNA lesions on template. Cell recognizes such a replication fork block and starts to operate several cellular functions that overcome the replication fork block. It has been known that post-replicative recombination repair and SOS repair are induced upon DNA replication fork block in E.coli cells. Most recently, these functions appeared to be operating also in eukaryotic cells. Aim of this project was to clarify molecular mechanisms of translesion DNA synthesis in E.coli and higher eukaryote.1.Assay for mutagenesis in in vitro DNA replication using E.coli replicative apparatus : We have established several systems for DNA replication in vitro reconstituted from purified replicative enzymes and succeeded in determining forward mutations caused by the DNA replication. It appeared that base substitution mutations were induced at several hot spots by DNA replication errors resulting from spontaneous DNA damages. Furthermore, the most frequent replication errors by the replicative apparatus were found to be those leading to single base frameshifts. This type of replication errors were highly specifically made at runs of same nucleotides in the template sequence.2.Analyses of natural pausing sites for DNA chain elongation using in vitro DNA replication system : We investigated pausing sites for DNA chain elongation by DNA polymerase III holoenzyme of E.coli using in vitro system with M13 single-stranded phage DNA as template. 8 strong pausing regions were found, and each of them consists of several pausing sites. About half of the pausing sites are able to form stable hair-pin structures.3.Identification of factors involved in translesion DNA synthesis in Xenopus laebis oocyte extracts : Using synthetic oligomer DNA that contains DNA lesion at a particular site, we have searched enzymaticactivities for translesion DNA synthesis. At least three different pathways were found.