Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York, United States of America.
PLoS Pathog. 2010 Jul 8;6(7):e1000992. doi: 10.1371/journal.ppat.1000992.
Homologous recombination (HR) mediates one of the major mechanisms of trypanosome antigenic variation by placing a different variant surface glycoprotein (VSG) gene under the control of the active expression site (ES). It is believed that the majority of VSG switching events occur by duplicative gene conversion, but only a few DNA repair genes that are central to HR have been assigned a role in this process. Gene conversion events that are associated with crossover are rarely seen in VSG switching, similar to mitotic HR. In other organisms, TOPO3alpha (Top3 in yeasts), a type IA topoisomerase, is part of a complex that is involved in the suppression of crossovers. We therefore asked whether a related mechanism might suppress VSG recombination. Using a set of reliable recombination and switching assays that could score individual switching mechanisms, we discovered that TOPO3alpha function is conserved in Trypanosoma brucei and that TOPO3alpha plays a critical role in antigenic switching. Switching frequency increased 10-40-fold in the absence of TOPO3alpha and this hyper-switching phenotype required RAD51. Moreover, the preference of 70-bp repeats for VSG recombination was mitigated, while homology regions elsewhere in ES were highly favored, in the absence of TOPO3alpha. Our data suggest that TOPO3alpha may remove undesirable recombination intermediates constantly arising between active and silent ESs, thereby balancing ES integrity against VSG recombination.
同源重组（HR）通过将不同的变异表面糖蛋白（VSG）基因置于活性表达位点（ES）的控制之下，介导锥虫抗原变异的主要机制之一。据信，大多数 VSG 转换事件是通过复制性基因转换发生的，但只有少数与 HR 密切相关的 DNA 修复基因被认为在该过程中起作用。与交叉相关的基因转换事件在 VSG 转换中很少见，类似于有丝分裂 HR。在其他生物体中，TOPO3alpha（酵母中的 Top3），一种 I 型拓扑异构酶，是参与抑制交叉的复合物的一部分。因此，我们询问是否存在相关机制可以抑制 VSG 重组。使用一组可靠的重组和转换测定法，可以对单个转换机制进行评分，我们发现 TOPO3alpha 功能在布氏锥虫中是保守的，并且 TOPO3alpha 在抗原转换中起着关键作用。在缺乏 TOPO3alpha 的情况下，转换频率增加了 10-40 倍，这种超转换表型需要 RAD51。此外，在缺乏 TOPO3alpha 的情况下，70-bp 重复序列对 VSG 重组的偏好减轻，而 ES 中其他地方的同源区域则受到高度青睐。我们的数据表明，TOPO3alpha 可能会不断去除活跃和沉默 ES 之间产生的不需要的重组中间体，从而在 ES 完整性与 VSG 重组之间保持平衡。
