Molecular mechanisms generating and suppressing spontaneous mutations

点击翻译按钮获取中文摘要

Spontaneous mutations are derived from various sources, including errors made during replication of undamaged template DNA, mutagenic nucleotide substrates, and endogenous DNA lesions. These sources vary in their frequencies and resultant mutations, and are differently affected by the DNA sequence, DNA transactions, and cellular metabolism. Organisms possess a variety of cellular functions to suppress spontaneous mutagenesis, and the specificity and effectiveness of each function strongly affect the pattern of spontaneous mutations. Base substitutions and single-base frameshifts, two major classes of spontaneous mutations, occur non-randomly throughout the genome. Within target DNA sequences there are hotspots for particular types of spontaneous mutations; outside of the hotspots, spontaneous mutations occur more randomly and much less frequently. Hotspot mutations are attributable more to endogenous DNA lesions than to replication errors. Recently, a novel class of mutagenic pathway t … More hat depends on short inverted repeats was identified as another important source of hotspot mutagenesis. In our project, we focused on molecular mechanisms that induce and suppress the spontaneous mutagenesis. From systematic analyses of spontaneous mutations occurring in wild-type and mismatch-repair deficient mutant of Eschrichia coli as well as mice, we first revealed that almost all of the DNA replication errors are corrected by the mismatch repair system and do not contribute to the generation of spontaneous mutations. Therefore, most of the spontaneous mutations are likely to arise from pre-mutagenic DNA lesions that are resistant to the mismatch-repair correction. As a major source of such pre-mutagenic lesion, we examined relative contribution of spontaneous oxidative DNA lesion and found that hydroxyl radicals are responsible to produce spontaneous base substitutions, especially hot-spot type of mutations. As another source of spontaneous mutations, we identified templete-switching type of DNA replication errors. This involves inverted repeats in geenome DNA, and processing of Okazaki-fragment seemed to play a crucial role. Furthermore, it appeared that exonuclease I sharply suppresses spontaneous mutagenesis caused by the template-switching errors. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. Less