Cerutti H, Johnson A M, Boynton J E, Gillham N W
Department of Botany, Duke University, Durham, North Carolina 27708, USA.
Mol Cell Biol. 1995 Jun;15(6):3003-11. doi: 10.1128/MCB.15.6.3003.
The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.
叶绿体中同源DNA重组的发生已有充分记录，但对于其中涉及的分子机制及其生物学意义却知之甚少。质体的内共生起源以及最近发现的一个拟南芥核基因，该基因编码一种与大肠杆菌RecA同源的叶绿体定位蛋白，这表明质体重组系统与其真细菌对应物有关。因此，我们研究了大肠杆菌RecA蛋白的显性负突变体是否会干扰单细胞绿藻莱茵衣藻叶绿体中其假定同源物的活性。表达这些突变RecA蛋白的转化体在暴露于DNA损伤剂时存活率降低，叶绿体DNA修复缺陷，质体DNA重组减少。这些结果有力地支持了叶绿体中存在RecA介导的重组系统。我们还发现，野生型大肠杆菌RecA蛋白可使质体DNA重组频率提高15倍以上，尽管它对DNA修复或细胞存活没有影响。因此，叶绿体DNA重组似乎受链交换相关酶可用性的限制，而非起始DNA底物水平的限制。我们的观察结果表明，质体中重组系统的主要生物学作用是修复其DNA，这很可能是应对光氧化和其他环境胁迫造成的损伤所必需的。这一假说可以解释尽管叶绿体基因组主要是单亲遗传，但DNA重组在叶绿体中仍具有进化保守性的现象。
