Molecular mechanisms of mutagenesis caused by transletion DNA synthesis

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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.