Scientific Literacy Paper

The circadian clock ensures successful DNA replication in cyanobacteria

  Synechococcus is a genus of cyanobacteria found throughout marine and freshwater environments.  These unicellular bacteria play a significant role in primary production, being responsible for approximately 25% of production (MicrobeWiki,2011).  Synechococcus elongatus is a gram-negative species of cyanobacteria that has been studied extensively and its genome mapped to be used for scientific research purposes.

  Synechococcus elongatus is an oligotrophic freshwater species (MicrobeWiki, 2011). It has the capabilities of surviving and thriving in extreme environments with unfavorable conditions such as light variability, high salinity, and fluctuations in temperature and CO₂ levels (Jaiswal et al, 2018). The structure of S. elongatus is composed of a cell envelope that is made of a thin peptidoglycan cell wall between an inner and outer cell membrane (Wikipedia Contributors, 2021).  Inside the cell membrane are several thylakoid membranes where photosynthesis takes place and phosphate bodies and carboxysomes in the cytoplasm (Jaiswal et al, 2018). The genetic make-up consists of a single circular chromosome and 2 plasmids.  The outer surface lacks flagellum or cilia so instead these bacteria move with a wave-like motion.  Their movement is not influenced by light availability as well (MicrobeWiki, 2011).

  S. elongatus is a photoautotroph with its own circadian rhythm that has important implications in studying DNA replication.  It undergoes oxygenic photosynthesis as its means of cellular respiration.   It acquires energy from the sun and uses CO² as its carbon source.  As mentioned previously, photosynthesis occurs within the thylakoid membranes that house the photosynthetic pigments, mostly chlorophyll a along with accessory pigments like phycobiliproteins.  Water is used to donate electrons which releases oxygen as a by-product and then the carbon dioxide is processed into glucose via the Calvin cycle (MicrobeWiki, 2011).

  Several species of Synechococcus have had their genomes mapped.  S. elongatus has been used in models to study the circadian rhythm and its impact on DNA replication.  Two of the factors used in DNA replication are a protein complex called beta-clamp (β-clamp) and single-stranded DNA binding protein (SSB).  The β-clamp protein binds to DNA polymerase and prevents it from dissociating from the template DNA strand.  It can also increase the rate of DNA synthesis because of its association with the rate limiting step (Wikipedia Contributors, 2020). The β-clamp dramatically increases the number of nucleotides the polymerase can add to the growing strand, thus increasing the rate of DNA synthesis up to 1,000-fold compared with a non-processive polymerase (Wikipedia Contributors, 2020).

  The SSB protein is a dimer composed of 3 beta-strands linked to a single six-stranded beta sheet (Wikipedia, 2020). It is a protein that binds to single-stranded regions of DNA and modulates the functions of proteins during the processes of DNA synthesis like replication, recombination, and repair. As DNA replicates, single strands are at a greater risk for degradation which can cause mutations that lead to things like cancer or can stall the replication process which causes further problems.  To protect these strands, a SSB protein essentially coats and protects the strand from unwanted deviations and can also increase the rate of the processes in correct conditions (Maffeo and Aksimentiev,2017).

  By capitalizing on the properties of Synechococcus elongatus as a photosynthetic organism with its own circadian rhythm, scientists have been able to study and manipulate the affects that altering light exposure has on its DNA replication.  The experiment consisted of studying wild type (WT) S. elongatus and genetically modified versions of the same bacteria in an environment where they were set on a clock of alternating 12-hour light and 12-hour dark periods for 48 hours.  This was done to regulate and synchronize their circadian patterns.  The cells were then transferred to an all-light situation.  Once transferred, the cells were infused with a fluorescent marker that could be used to monitor DNA replication by correlating the area of fluorescence as active replication (Liao and Rust, 2020).  It was found that there was a surge of activity shortly after “dawn” or when the light stage was engaged, and activity gradually tapered down as evident in Figures 2A and 2B.  There were peaks at approximately 18 hours, 42 hours, and 60 hours which can be seen by the presence of fluorescence and troughs at the 30 and 58 hours.  It was concluded that in constant conditions, peak replication took place at subjective dawn and trough was near subjective dusk.

  Not only does light have an affect on the timing of replication, but also the rate.  The presence of β-clamp and SSB proteins were studied and charted in response to not only the light and dark cycles, but also the intensity of the light.  As stated above, β-clamp and SSB are important components that speed up the rate of DNA replication.  On its own, S. elongatus can grow with a wide range of doubling times and hence time needed to replicate based on the intensity of light exposure.  Figure 2D and 2E showed that the doubling time was clearly affected by the intensity of the light, where the highest intensity showed a more rapid doubling time compared to the others, but the concentrations of β-clamp and SSB showed little variance which led to the conclusion that the replication process is more dependent on the initiation time not the abundance of the proteins (Liao and Rust, 2020).

  The presence of active replication does not necessarily mean active cell division in all cases.  However, this set of experiments did establish the correlation that DNA replication is tied to bacterial cell division by studying the effects of removing genes such as KaiBC that have a role in the circadian rhythm response.  This strain of bacteria had this gene deleted and thus over time there was no cycling to produce thymin and thus no replication.  Now that it was established that replication and division is dependent on normal light/dark cycles, the experiment continued to address the effects of disrupting those cycles and how it impacted the replication process.  It was concluded that incorrect clock states such as introducing night in the middle of an anticipated light cycle would cause the replication process to abort.  However, the experiments also found that by reintroducing light to either in-phase or out-of-phase organisms, they were able to resume replication and division, albeit with some minor changes such as cell growth being mildly stunted compared to other uninterrupted cells.

  This set of experiments did establish a correlation between circadian rhythms and replication by finding that cells will work to complete all replication processes prior to nightfall when energy reserves are low and avoid having to abort those processes.  Circadian rhythms are a fundamental part to many areas of biophysiology from the minute but critical DNA replication to sleep schedules.

References

1. https://microbewiki.kenyon.edu/index.php/Synechococcus_elongatus
2. Jaiswal, D., Sengupta, A., Sohoni, S. et al. Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India. Sci Rep 8, 16632 (2018). https://doi.org/10.1038/s41598-018-34872-z
3. Wikipedia contributors. (2021). Gram-negative bacteria, https://en.wikipedia.org/wiki/Gram-negative_bacteria
4. Wikipedia contributors. (2020). DNA clamp, https://en.wikipedia.org/wiki/DNA_clamp Wikipedia contributors. (2020). Single-strand DNA binding protein, https://en.wikipedia.org/wiki/Single-strand_DNA-binding_protein
5. Maffeo, Christopher and Aksimentiev, Aleksei (2017). Molecular mechanism of DNA association with single-stranded DNA binding protein. Nucleic Acids Rresearch, 45(21): 12125–12139. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716091/
6. Liaoa, Yi and Rusta, Michael J. The circadian clock ensures successful DNA replication in cyanobacteria.