Molecular mechanisms generating and suppressing spontaneous mutations

自发突变有多种来源，包括在复制未受损的模板DNA、突变的核苷酸底物和内源性DNA损伤过程中出现的错误。这些来源的频率和所产生的突变各不相同，并且受到DNA序列、DNA交易和细胞代谢的不同影响。生物体具有多种抑制自发突变的细胞功能，而每种功能的特异性和有效性强烈影响着自发突变的模式。碱基替换和单碱基移码是两类主要的自发突变，在整个基因组中非随机发生。在靶DNA序列中，有特定类型的自发突变的热点；在热点之外，自发突变发生的概率更高，频率要低得多。热点突变更多地归因于内源性DNA损伤，而不是复制错误。最近，一类新的诱变途径t…更多的HAT依赖于短反向重复序列被认为是热点突变的另一个重要来源。在我们的项目中，我们重点研究了诱导和抑制自发突变的分子机制。通过对大肠杆菌野生型和错配修复缺陷突变体以及小鼠自发突变的系统分析，我们首次揭示了几乎所有的DNA复制错误都可以被错配修复系统纠正，并不有助于自发突变的产生。因此，大多数自发突变很可能产生于对错配修复纠正具有抵抗力的前突变DNA损伤。作为这种诱变前损伤的主要来源，我们检查了自发性氧化DNA损伤的相对贡献，发现羟基自由基是导致自发碱基替换的原因，特别是热点类型的突变。作为自发突变的另一个来源，我们发现了模板切换型DNA复制错误。这涉及到基因组DNA中的反向重复，而冈崎片段的处理似乎起到了关键作用。此外，核酸外切酶I似乎能显著抑制模板切换错误引起的自发突变。使用一个半双向DNA复制系统，该系统使用纯化的复制酶和大肠杆菌的复制终止蛋白对ORIC质粒进行半双向DNA复制，当它遇到模板DNA上的单个基本DNA损伤时，我们检测了复制叉的动态。位于滞后链上的DNA损伤完全阻止了延伸到病变部位的冈崎片段的合成，但不影响复制叉或前导链DNA合成的进展。相比之下，前导链上的DNA损伤使复制分叉停滞，同时强烈抑制前导链合成。然而，大约三分之二遇到这种病变的复制叉子在病变部位之外保持了约1kb的滞后链合成，复制叉子前进的速度似乎显著降低。较少

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