Intracellular Organization: Getting ready for DNA duplication
The discovery of a biomolecular condensate involved in DNA replication has wide ranging implications.
- The Rockefeller University, United States
Research into biomolecular condensates – self-contained regions within cells where specific reactions take place – is taking cell biology by storm. Biomolecular condensates do not have membranes, but they are able to keep the proteins and/or nucleic acids involved in a particular reaction separate from the rest of the cell. Biomolecular condensates do not have membranes, but they are able to keep the molecules involved in a particular reaction (usually proteins, but sometimes also nucleic acids) separate from the rest of the cell (Boeynaems et al., 2018; Ditlev et al., 2018). They can be found in both the nucleus and the cytoplasm, and are involved in reactions ranging from signal transduction to RNA metabolism. The nucleolus, a structure in the nucleus where ribosomes are synthesized, is an example of a biomolecular condensate (Ahmad et al., 2009).
The physics behind the formation of biomolecular condensates is not fully understood but it often involves at least one protein with an intrinsically disordered region that does not fold into a specific 3D structure, and a process called phase separation (which, as its name suggests, involves regions with different physical properties separating from each other). The molecules required for the formation of condensates are called scaffolding factors, while the molecules encapsulated inside are known as clients. A given condensate typically contains only those clients involved in the relevant reaction.
Now, in eLife, Michael Botchan (UC Berkeley), James Berger (Johns Hopkins) and colleagues – including Matthew Parker as first author – report on the discovery of a biomolecular condensate required for the initiation of DNA replication in the fruit fly Drosophila melanogaster (Parker et al., 2019). Three proteins – ORC, Cdc6 and Cdt1 – form the scaffold along with DNA, while a hexamer ring protein called Mcm2-7 is the client. During the G1 phase of the cell cycle, the scaffold proteins load two Mcm2-7 hexamers onto the DNA to form the pre-replicative complex, which marks the spot where DNA replication will begin (Figure 1A–C; Bell and Labib, 2016; Bleichert et al., 2017). Later, in the S phase of the cell cycle, other proteins assemble onto the complex, converting the Mcm2-7 hexamers into helicases that unwind the DNA for replication (Figure 1D).
Figure 1
The molecules involved in the initiator biomolecular condensate during the G1 and S phases of the cell cycle.
(A) Parker et al. studied how three proteins – ORC (green), Cdt1 (purple) and Cdc6 (blue) – work together to help two Mcm2-7 hexamers (yellow) form a pre-replicative complex with DNA (red). (B) During the G1 phase of the cell cycle intrinsically disordered regions (IDR) in the origin of replication complex (ORC), Cdc6 and Cdt1 bind a DNA molecule to form a biomolecular condensate. (C) The biomolecular condensate forms a scaffold that the Mcm2-7 hexamer can enter, and the pre-replicative complex is formed. (D) During the S phase of the cell cycle, the IDRs of the scaffold proteins are phosphorylated (represented by the letter P), so they can no longer form condensates. Mcm2-7 hexamers loaded on the DNA can then interact with other proteins (orange and yellow) to form helicases that unwind the DNA during replication.
When Parker et al. mixed Drosophila Cdt1 with DNA, they obtained a turbid solution that formed oil-like droplets under the microscope, a hallmark of biomolecular condensates. The same effect was observed when they mixed Drosophila ORC and Cdc6 with DNA. Additionally, Parker et al. found that they could disrupt the formation of these condensates by deleting the intrinsically disordered regions of any of the three proteins involved. On the other hand, Mcm2-7 failed to form a condensate when mixed with DNA. Instead, it acted as a client that could enter the ‘initiator biomolecular condensate’ formed by the scaffold proteins, and once inside, be loaded onto the DNA. Parker et al. demonstrated the selectivity of this condensate by showing that it denied entry to a protein that can form other biomolecular condensates but is not involved in initiating DNA replication.
The initiator biomolecular condensate is required for DNA replication to start, an important step in the cell cycle. Parker et al. found that Cdc6, Cdt1 and ORC could all be phosphorylated on their intrinsically disordered regions by cell cycle kinases, which stopped the formation of the condensate. This interaction between cell cycle kinases and the scaffold proteins links the formation of the pre-replicative complex to the cell cycle.
But why would the cell form a biomolecular condensate for the pre-replicative complex reaction at all? Parker et al. note that the budding yeast, a cell with a relatively small genome, does not form an initiator condensate. Hence, the selection pressure to evolve this particular condensate might lie in the genomic complexity of organisms.
When animal cells divide, DNA duplication starts at specific sites in the genome called origins of replication, where the pre-replicative complexes are assembled. There are multiple origins of replication throughout the genome, and the start of DNA duplication at each one is referred to as ‘firing’. Origins of replication appear to fire stochastically, with some firing early during the S phase of the cell cycle and others later on (Kaykov and Nurse, 2015). But if one takes an eagle’s view, there are indications that the process may happen in a particular order in time and space. For example, origins that fire early sometimes appear in clusters, and certain origins prevent others from firing, suggesting that the origins can communicate with each other (Cayrou et al., 2011; Duriez et al., 2019). Thus, initiator biomolecular condensates might gather distant regions of DNA to form pre-replicative complexes in a cluster, facilitating temporal and spatial organization of replication start sites. The discovery of these condensates therefore represents an important step toward a more complete understanding of DNA replication in complex genomes.
References
-
NOPdb: Nucleolar Proteome Database--2008 updateNucleic Acids Research 37:D181–D184.https://doi.org/10.1093/nar/gkn804
-
Protein phase separation: a new phase in cell biologyTrends in Cell Biology 28:420–435.https://doi.org/10.1016/j.tcb.2018.02.004
-
Who's in and who's out-Compositional control of biomolecular condensatesJournal of Molecular Biology 430:4666–4684.https://doi.org/10.1016/j.jmb.2018.08.003
Article and author information
Author details
Michael E O'Donnell
Publication history
Copyright
© 2019, Yao and O'Donnell
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,781
- views
-
- 195
- downloads
-
- 3
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Intracellular Organization: Getting ready for DNA duplication
eLife 8:e51291.
https://doi.org/10.7554/eLife.51291
Further reading
-
- Biochemistry and Chemical Biology
In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to approximate compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
-
- Biochemistry and Chemical Biology
The intestinal absorption of essential nutrients, especially those not readily biosynthesized, is a critical physiological process for maintaining homeostasis. Numerous studies have indicated that intestinal absorption is mediated by various membrane transporters. Citrate, a crucial bioactive compound produced as an intermediate in the Krebs cycle, is absorbed in the small intestine through carrier-mediated systems because of its high hydrophilicity. While the luminal absorption of citrate is mediated by Na+-dicarboxylate cotransporter 1 (NaDC1/SLC13A2), the mechanism governing the release of the transported citrate into the bloodstream remains unknown. Here, we explored the transporters responsible for intestinal citrate absorption at the basolateral membrane, focusing on highly expressed orphan transporters in the small intestine as candidates. Consequently, SLC35G1, originally identified as a partner of stromal interaction molecule 1, a cell surface transmembrane glycoprotein, was found to play a role in the intestinal absorption of citrate at the basolateral membrane. Furthermore, our results revealed that SLC35G1-mediated citrate transport was diminished by chloride ions at physiologically relevant extracellular concentrations. This suggests that SLC35G1, to our best knowledge, is the first transporter identified to be extremely sensitive to chloride ions among those functioning on the basolateral membrane of intestinal epithelial cells. This study provides valuable insights into the intestinal absorption of citrate and significantly contributes to elucidating the poorly understood molecular basis of the intestinal absorption system.
