Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculate
Reproduction, the hallmark of biological activity, requires making an accurate copy of the genetic material to allow the progeny to inherit parental traits. In all living cells, the process of DNA replication is carried out by a concerted action of multiple protein species forming a loose protein–nucleic acid complex, the replisome. Proofreading and error correction generally accompany replication but also occur independently, safeguarding genetic information through all phases of the cell cycle. Advances in biochemical characterization of intracellular processes, proteomics, and the advent of single‐molecule biophysics have brought about a treasure trove of information awaiting to be assembled into an accurate mechanistic model of the DNA replication process. This review describes recent efforts to model elements of DNA replication and repair processes using computer simulations, an approach that has gained immense popularity in many areas of molecular biophysics but has yet to become mainstream in the DNA metabolism community. It highlights the use of diverse computational methods to address specific problems of the fields and discusses unexplored possibilities that lie ahead for the computational approaches in these areas.
more » « less- NSF-PAR ID:
- 10461249
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Theory and Simulations
- Volume:
- 2
- Issue:
- 5
- ISSN:
- 2513-0390
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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in silico chromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM. -
The DNA inside human cells provides instructions for all of the processes that happen inside the body. Errors in the DNA may lead to cancer, sickle cell disease, cystic fibrosis, Huntington’s disease, or other genetic disorders. Medical researchers are exploring whether it is possible to replace or repair the faulty DNA (an approach known as gene therapy) to reduce the symptoms, or even cure individuals, of these conditions. Over the last ten years, a new technology known as CRISPR-Cas9 gene editing has proved to be a reliable and efficient way to make small and precise changes to DNA in living cells. First, an enzyme called Cas9 searches for a segment of target DNA segment that matches a template molecule the enzyme carries. Cas9 then cuts the target DNA, which is repaired to match a new customized DNA sequence: this changes the genetic information of the cell. The Cas9 protein is made of a succession of building blocks called amino acids that create long chains which then fold to form the final three-dimensional shape of the enzyme. A region of Cas9 known as the HNH domain is responsible for cutting the target DNA. However, it remains unclear exactly which amino acids within this domain work together to sever the DNA. Here, Zuo et al. combined computational and experimental approaches to reveal the three-dimensional structure of the Cas9 enzyme when the HNH domain is poised to cut the target DNA. The findings were used to generate a computational model of Cas9 and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands. This knowledge is useful to understand exactly how Cas9 modifies genetic information. Ultimately, this may help to improve CRISPR-Cas9 technology so it could be safely used in geneediting therapies.more » « less
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Summary Bacteria coordinate DNA replication and cell division, ensuring a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage, a cell cycle checkpoint is activated by expressing a cell division inhibitor. The prevailing model is that activation of the DNA damage response and protease‐mediated degradation of the inhibitor is sufficient to regulate the checkpoint process. Our recent genome‐wide screens identified the gene
ddcA as critical for surviving exposure to DNA damage. Similar to the checkpoint recovery proteases, the DNA damage sensitivity resulting fromddcA deletion depends on the checkpoint enforcement protein YneA. Using several genetic approaches, we show that DdcA function is distinct from the checkpoint recovery process. Deletion ofddcA resulted in sensitivity toyneA overexpression independent of YneA protein levels and stability, further supporting the conclusion that DdcA regulates YneA independent of proteolysis. Using a functional GFP‐YneA fusion we found that DdcA prevents YneA‐dependent cell elongation independent of YneA localization. Together, our results suggest that DdcA acts by helping to set a threshold of YneA required to establish the cell cycle checkpoint, uncovering a new regulatory step controlling activation of the DNA damage checkpoint inBacillus subtilis . -
null (Ed.)Abstract Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.more » « less
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