Lee Po-Hsuen, Meng Xiangzhou, Kapler Geoffrey M
Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center, College Station, Texas, United States of America.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.
PLoS Genet. 2015 Jan 8;11(1):e1004875. doi: 10.1371/journal.pgen.1004875. eCollection 2015 Jan.
The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.
由于单个细胞质中存在两个功能不同的细胞核以及复杂的发育程序，嗜热四膜虫的DNA复制机制面临着独特的需求。在此，我们提供了ORC和MCM丰度发生程序性变化的证据，这些变化与传统的DNA复制模型不一致。作为起点，我们表明ORC剂量在营养细胞周期和发育过程中至关重要。Orc1p适度减少会导致二倍体微核中的基因组不稳定、多倍体大核的异常分裂以及无法产生强大的S期内检查点反应。与酵母ORC2突变体不同，复制起始不受影响；相反，随着Mcm6p水平与Orc1p平行下降，复制叉延伸受到干扰。实验诱导的ORC和MCMs下调也会损害核内复制和基因扩增，这与它们在发育过程中的重要作用一致。出乎意料的是，在野生型接合体发育过程中，Orc1p和Mcm6p水平剧烈波动，在传统微核DNA复制和大核原基复制（核内复制I期，rDNA基因扩增）的早期周期中增加。这种增加并不反映DNA复制负荷，因为与营养S期相比，在此发育窗口期间合成的DNA要少得多。此外，尽管Orc1p水平在核内复制II期之前短暂增加，但当复制负荷增加并产生非常规DNA复制中间体时，Orc1p和Mcm6p水平会下降。我们提出，在嗜热四膜虫发育的连续阶段，复制起始被重新编程以满足不同的需求或挑战。
