Institute of Molecular and Cellular Biology SB RAS Novosibirsk, Novosibirsk, Russia.
Novosibirsk State University, Novosibirsk, Russia.
Chromosome Res. 2022 Dec;30(4):361-383. doi: 10.1007/s10577-021-09679-w. Epub 2022 Feb 28.
Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.
研究染色体复制起始的概率分布是一个巨大的挑战。结合超分辨率显微镜的果蝇多线染色体为在超微结构水平上分析复制起始的概率性质提供了独特的机会。在这里，我们开发了一种同步唾液腺细胞 S 期诱导的方法。对 S 期诱导后最初几分钟和随后几个小时的复制标记分布的分析揭示了复制起始的动力学。空间超分辨率结构照明显微镜允许识别多个离散的复制信号，并在超微结构水平上研究 S 期最初几分钟的复制信号的行为。我们确定了复制起始区，其中起始是随机发生的。这些区域在每个时间单位的复制起始概率上有显著差异。在一些区域，几分钟内多线染色体的大多数链上都会发生起始。在其他区域，所有链上的起始都需要几个小时。致密带中没有复制起始事件，复制从外边缘到中间进行，在此过程中带的形状可能会发生改变。
