This award supports research focused on understanding the role of important proteins in the structure and function of a bacterial cell. The size of the DNA requires that it be compacted to fit the dimensions of the cell, while still allowing its accessibility to cellular proteins. The bacterial genome is associated with a number of different DNA-binding proteins, including proteins known as HU. Aside from being involved in the organization in DNA, HU proteins function in regulating DNA-dependent processes, such as replication, repair and gene expression and are therefore critical for normal cellular function. In this project, the focus will be on the HU variant encoded by Mycobacterium smegmatis. Mycobacteria encode an unusual two-domain HU protein. What is intriguing about two-domain HU proteins (typically referred to as Hlp, for Histone Like Protein) is that they appear to be encoded only by bacterial species that are particularly adept at resisting environmental stress. For mycobacteria, Hlp has, for example, been reported to be more abundant during dormancy. The goal of this research is to define the functional role of M. smegmatis Hlp, with a specific focus on the role of the lysine-rich extension. Understanding functional roles of mycobacterial Hlp will illuminate a unique aspect of mycobacterial physiology as well as the specific demands of other bacterial species that encode two-domain HU homologs. A combination of DNA-binding assays designed to determine the role of Hlp in DNA compaction are planned, as well as assays aimed at determining the ability of Hlp to protect DNA from damage. M. smegmatis also encodes a protein named Ku, which is expected to function in repair of DNA double strand breaks; Ku shares with Hlp key features of the lysine-rich extension. This shared sequence feature will be exploited to determine the role of the lysine-rich domain in DNA end-joining by both proteins and to ascertain their roles in double strand break repair. A final aim is centered on determining conditions under which Hlp is more abundant and on investigating consequences of Hlp deletion on global gene expression. This line of inquiry is expected to define conditions under which excess Hlp is required and the roles of Hlp in DNA organization, DNA protection, and DNA double strand break repair. This project will integrate research and education by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with analysis of protein-protein and protein-nucleic acid interactions. The involvement of minority students will be emphasized, and students at all levels will be expected to disseminate their research at national meetings.
INTELLECTUAL MERIT: This project concerned mechanisms by which bacterial cells deal with environmental stress, particularly conditions with the potential to damage genomic DNA. Understanding such responses to environmental stress is important, for example in the context of bacterial species that function in bioremediation of waste sites or plant pathogens that experience inhospitable environments upon infecting their host. The bacterial genomic DNA is compacted and protected by association with a number of small basic proteins, forming a complex commonly referred-to as the nucleoid. One of the proteins that serve this function is named HU. In Mycobacteria, the corresponding protein, which is named Hlp, has an N-terminal domain that resembles conventional HU proteins and an unusual C-terminal extension composed of numerous short sequence repeats consisting of the amino acids proline, alanine, and lysine. In general, Hlp proteins with such C-terminal sequence repeats are encoded by bacterial species that are more resistant to environmental stress. For example, they serve a role in dormancy, a non-replicating, but viable state that is characteristic of Mycobacteria. The genomic DNA may suffer double strand breaks as a consequence of exposure to environmental stress. One of the repair pathways responsible for repair of such lesions in Mycobacteria is non-homologous end-joining in which the broken DNA ends are brought into close proximity and subsequently ligated together. The protein that is responsible for bringing the DNA ends together is Ku, a ring-shaped molecule that binds DNA ends. Notably, Ku encoded by environmental mycobacterial species such as M. smegmatis contain a C-terminal extension characterized by the same proline-, alanine-, and lysine-containing repeats found in Hlp. A main objective of the project was to understand the functional significance of these lysine-rich sequence repeats. The salient observations are: (1) The lysine-rich C-terminal extensions of M. smegmatis Hlp and Ku confer on the proteins the ability to bring together DNA ends or promote DNA compaction. This property would be predicted to facilitate repair of DNA double strand breaks by Ku and to promote protection of genomic DNA by Hlp, in both circumstances enhancing protein function and contributing to stress resistance. Since our findings point to a common function of the lysine-rich repeats in DNA association, other DNA-binding proteins that contain similar repeats may likewise participate in DNA association. (2) M. smegmatis Ku binds DNA ends, following which it translocates to internal DNA sites. Such movement on the DNA, which has not been reported for other bacterial Ku proteins, is reminiscent of the function of eukaryotic Ku and may facilitate access to the cognate DNA ligase that is responsible for joining DNA ends. (3) M. smegmatis Ku can bind internal DNA sites directly by means of its lysine-rich C-terminal extension. Since such binding would increase the concentration of Ku in the vicinity of DNA, it may facilitate recruitment to damaged DNA. (4) M. smegmatis Ku binds zinc. Such zinc binding has little effect on DNA binding. Instead, it confers a tolerance to toxic levels of zinc, and the gene encoding Ku is upregulated in presence of zinc. We have therefore proposed a novel moonlighting function of Ku in sequestering excess zinc without compromising its function in DNA repair. (5) The gene encoding Hlp is upregulated by iron, and the protein is capable of converting Fe2+ to Fe3+; the significance of this observation is that Hlp may reduce formation of reactive hydroxyl radicals that may damage cellular components. Taken together, we have established that two DNA-binding proteins that play important roles in mycobacterial physiology have attained novel or enhanced functions that are consistent with enhanced responses to environmental stress. BROADER IMPACTS: Broader impacts of this award have centered on student training. Several graduate students received training under this award, which has resulted in the completion of three Ph.D. dissertations and one Masters thesis. Upon graduation, all Ph.D. students had published at least two first-author research articles and presented their research at several national meetings; two accepted positions as postdoctoral researchers after graduation, and one pursued a career in teaching. One postdoctoral research scientist was also trained under this award, and two high school students completed research projects in the laboratory. A total of 16 undergraduate students, including five members of underrepresented groups, participated in research; most have graduated and enrolled either in graduate school or medical school. One undergraduate student was a co-author on a research article, and several presented their research at national meetings.
