Biophysical and Photochemical studies of Nucleoid Associated Proteins with special emphasis on HU and Dps

Abstract

Eukaryotic DNA compaction is mediated by histones and their variants, with abundant literature surrounding role of Liquid-Liquid phase separation of histones and how the same aids in DNA compaction and regulation. In stark contrast, the homologous mechanisms in relatively 'simpler' prokaryotes are yet to be fully elucidated. Prokaryotes, like the standard model organism E. coli, lack histones but possess Nucleoid Associated Proteins (NAPs) responsible for functions similar to histones. It has been hypothesised, and proven, that the bacterial nucleoid exists in a state of phase separation from the surrounding cytoplasm although the mechanism has remained a mystery. In Part A of this thesis, we shed light on the mechanism of E. coli nucleoid phase separation by NAPs and its regulation. In the end of Part A we have shown how phase separation of NAPs outside E. coli can benefit in the process of biofilm formation. Moving on to Part B, we explore an intriguing observation—basic DNA compaction proteins lack tryptophan in their sequence. Our data reveals that indeed basic DNA compaction proteins from various species (both prokaryotic and eukaryotic) lack Tryptophan. Moreover, when a Trp-containing engineered version of these proteins is introduced, it can cause significant disruptions to the proteins themselves and the genomes of cells overexpressing the Trp-containing protein. In Chapter 1 and Chapter 2, general introduction and material methods, respectively, for this thesis are provided. Chapter 3 consists of the results and is subdivided into Part A and Part B, which have been further subdivided into multiple sections. Part A is titled as: Liquid-Liquid phase separation of HU and Dps with negatively charged molecules such as DNA and LPS. In A.1, an introduction is provided to various concepts associated with this section, such as Phase separation and Biofilm formation. In A.2 material and method used specifically in this section are provided. In A.3, we show that HU undergoes spontaneous complex coacervation (via Liquid-Liquid phase separation) when mixed with DNA, at entirely physiological concentrations and conditions. The resulting condensates are spherical and liquid like in nature. The molecular vdrivers for the formation of such conditions were also checked by varying different parameters, we found that electrostatic interactions were the primary drivers of the phenomenon. HU-A also showed complex coacervation with DNA, similar to HU-B but at a lower degree, it was verified that the reason for this was a difference in the multimeric nature of HU-A and HU-B. Interestingly, we observed that there exists a synergy between HU-B and HU-A, i.e., small amounts of HU-B are capable of phase separating the non-phase separating concentrations of HU-A when both the proteins are present in the same solution. We also verified may properties of HU-DNA condensates that should be compatible with phase separation of the nucleoid e.g., complex coacervation with DNA polymerase, complex coacervation with multiple different forms of DNA etc. In A.4, In A.4, we provide details for the cloning of the Nucleoid Associated protein called Dps. In this chapter we show that the protein was purified in our lab successfully and was folded properly. We confirmed that the protein is dodecameric and is DNA binding in nature, like it has been reported to be. Furthermore, we observed that Dps is well folded and dodecameric in the presence of physiological salt conditions. Below that, Dps loses its structure and most likely its multimericity as well. In A.5, we demonstrate that Dps is also capable of phase separating with DNA, similar to HU- A/B, forming highly dynamic condensates. The condensates formed are aberrantly shaped unlike the HU-DNA condensates, they appear to be spherical condensates that are stuck together but not fusing into a single structure. When Dps and HU-A/B are mixed together in presence of DNA, they form multiphasic heterotypic condensates in which HU condensates are enriched regions in rich with both DNA and Dps. Interestingly, biphasic heterotypic condensates are formed in absence of DNA. In A.6, we investigate the role of Liquid-Liquid phase separation of HU and Dps in biofilm formation. Biofilms consist of an extracellular matrix or Extracellular Polymeric Substance (EPS) that is rich in DNA and DNA binding proteins like HU, surprisingly, both of them are integral for the biofilm integrity. Phase separated condensates discussed in the previous sections are made of DNA and DNA binding proteins, which are essentially the same things of which biofilm EPS is made up of. We observed that E. coli cells get titrated towards HU-DNA condensates. HU and Dps can make undergo LLPS with LPS, in total absence of DNA, to make condensates that are spherical in nature but interestingly showed limited molecular diffusion virates. Upon addition of LPS to HU-DNA condensates the molecular diffusion rates drop down, presumably as mechanism to increase the viscosity of the EPS that could help in biofilm attachment. In A.7, we have shown cloning, purification and characterization of HU from thermophile called Themus thermophilus. We characterized the protein for its thermal and chemical stability. We observed that the HU from Themus thermophilus was also capable of phase separating similar to HU-A/B from E. coli. In B.1, Introduction to the photochemistry of Tryptophan photosensitization is provided. A hypothesis is fleshed out and explained that how DNA compaction proteins that are buried deep within DNA could get damaged by photosensitization of tryptophan. The photosensitiser containing DNA compaction protein could also damage the DNA bound to it, details regarding the possibility of the same are provided. In B.2, Material method specific to this section are provided. In B.3, Bioinformatics based sequence analysis was done to count the number of different amino acids in different DNA compaction proteins like NAPs of E. coli and histones of eukaryotes. We discovered that all the basic DNA compaction proteins like HU and Histones of almost all species, for which sequences were available, uniformly lacked tryptophan in their amino acid sequence. Then using a tryptophan containing mutant of HU-B (HU-B F47W), we show that such proteins and, not the Trp lacking wild type counterparts, are capable of creating the cascade of photosensitisers known to be created from photodegradation of Trp. Furthermore, all proteins studied in this thesis were getting crosslinked by UV-C but only proteins that had Trp in their sequence got crosslinked by UV-B. Notably, the HU-B F47W expressing cells showed significantly more damage to their genomes when irradiated with UV-B/A. In chapter 4, a conclusion to the entire thesis is provided. In chapter 5, a list of references to the literature that has been cited is provided.