Positioning and functional structure of repliction time zones at the cystic fibrosis gene locus

在前两个资助期间，我们研究了DNA复制的动态。我们可以证明，大规模的运动不参与DNA复制1，2。因此，我们集中在本地复制动力学的分析。结果显示，与优选的模型3-6相反，DNA以及复制蛋白质的功能复合物显示局部动力学。蛋白质复合物显示局部再定位，这不是由于整个复合物的运动，而是由于复制蛋白的组装和拆卸的局部过程。当准备复制的DNA可以被转移到这些复合物中时，新生的DNA以聚集的形式被挤出到相邻的区域中。此外，结果表明，93%的DNA聚集体，在S期的一个确定的时间点复制，稳定地保持其复制时间。这些结果有力地支持了这一假设，即染色体被组织成稳定的DNA聚集体（亚染色体焦点），其在S期7-13期间显示出确定的复制时间。为了进一步证明这一假设，我们将在下一个资助期内研究囊性纤维化基因位点14处的不同复制时区。我们将分析，这些不同的时区是否组织成稳定的亚染色体病灶显示一个明确的规定方面的复制，转录和核定位。

During the first two funding periods we investigated the dynamics of DNA replication. We could show, that large-scale movements are not involved in DNA replication 1,2. Therefore, we concentrated on the analysis of local replication dynamics. In results show that, in contrast to favored models 3-6, DNA as well as functional complexes of replication proteins display local dynamics. The protein complexes display local re-localizations, which are not due to movements of whole complexes, but due to local processes of assembly and disassembly of replication proteins. While the DNA poised to replicate can be translocated into these complexes, nascent DNA is extruded in an aggregated form into adjacent regions. Furthermore, the results showed that 93% of DNA aggregates, which replicated at a defined time point during S-phase, stably maintained their replication timing. These results strongly support the hypothesis, that chromosomes are organized into stable DNA aggregates (subchromosomal foci), which display a defined replication timing during S-phase 7-13. In order to further proof this hypothesis we will investigate the distinct replication time zones at the cystic fibrosis gene locus 14 during the next funding period. We will analyze, whether these distinct time zones are organized into stable subchromosomal foci displaying a defined regulation with regard to replication, transcription, and nuclear positioning.