染色体结构域在S期的异步复制对于真核生物基因组功能至关重要。但是,在不同细胞类型中,确定哪些结构域在早期和晚期复制的机制仍未完全了解。间位异染色质结构域在分裂细胞的二倍体染色体和内复制的多烯染色体中复制得非常晚,在这些染色体中它们也被低估了。果蝇SNF2相关因子SUUR赋予多烯染色体的基因座特异性复制不足。SUUR负调节DNA复制叉进程;然而,它的作用机制仍然模糊。在这里,我们开发了一种称为MS-Enabled快速蛋白质复合物鉴定(MERCI)的新方法,以分离包含SUUR和染色质边界蛋白Mod(Mdg4)-67.2的稳定化学计量天然复合物SUMM4。Mod(Mdg4)刺激SUURATP酶活性,是体内SUUR正常时空分布所必需的。SUUR和Mod(Mdg4)-67.2共同介导吉普赛绝缘子的活性,阻止某些增强子-启动子相互作用并在基因组中建立常染色质-异染色质屏障。此外,SuUR或mod(mdg4)突变可逆转间异染色质的复制不足。因此,SUMM4可以通过减弱复制叉通过常染色质/异染色质边界的进展,从而赋予间位异染色质的晚期复制。我们的发现暗示SNF2家族ATP依赖性运动蛋白SUUR在绝缘子功能中,揭示DNA复制可以被染色质屏障延迟,并揭示建筑蛋白在复制控制中的关键作用。他们提出了一种建立后期复制的机制,该机制不依赖于后期复制起点的异步触发。
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Inside cells, molecules of DNA provide the instructions needed to make proteins. Cells carefully maintain and repair their DNA, and typically make a complete copy of the genome before they divide to ensure that after division, each daughter cell has a full set. Within human, fly and other eukaryotic nuclei, DNA is packaged into structures known as chromosomes. Cells follow precisely controlled programs to replicate distinct regions of chromosomes at different times. To start copying a particular region, the cell machinery that replicates DNA binds to a sequence known as the origin of replication. It is thought that as-yet unknown cues from the cell may lead the replication machinery to bind to different origins of replication at different times. In some circumstances, cells make extra copies of their DNA without dividing. For example, many cells in the larvae of fruit flies contain hundreds of extra DNA copies to sustain their increased sizes. However, the entire genome is not copied during this process, so cells end up with more copies of some regions of the genome than others. A protein called SUUR is required for hindering the replication of the ‘underrepresented’ regions, but it is not clear how it works. To address this question, Andreyeva, Emelyanov et al. developed a new approach based on liquid chromatography and quantitative proteomics to identify the native form of SUUR in fruit flies. This revealed that SUUR exists as a stable complex with a protein called Mod(Mdg4), which is needed to recruit SUUR to the chromosomes. Further experiments suggested that SUUR and Mod(Mdg4) work together to bind to regions of DNA known as gypsy insulator elements, creating a physical barrier that hinders the replication machinery from accessing some parts of the genome. The findings of Andreyeva, Emelyanov et al. provide an alternative explanation for how individual cells may stagger the process of copying their DNA without relying on the replication machinery binding to various replication origins at different times. Rather, late replication timing may be instructed by an insulator-born delay of the progression of replication over particular genomic regions. This mechanism adds to the list of nuclear processes (chromosome partitioning, transcriptional regulation, etc.) that are known to be directed by insulators and associated architectural proteins.