DNA Binding Protein’s Structure, Mechanism, and Functional Relevance; From Filament Formation of SgrAI to Helicase Activity of B19V-NS1

Abstract

I have examined the structure, mechanism, and functional relevance of two DNA-binding proteins in this study. The first of these proteins is SgrAI. It is a type II restriction endonuclease that recognizes and cleaves certain sequences, known as primary and secondary sites. Depending on the type of recognition site SgrAI is bound to, SgrAI has a dimeric form or a filamentous form. The dimeric form of SgrAI is an inactive structure which can cleave only primary site sequences slowly. On the other hand, when it is in the filamentous form, its cleavage rate increases dramatically for both primary and secondary sites. In the absence of primary site sequences, SgrAI will not form filaments. In addition, by substituting the first or second base pair of the primary recognition site, the secondary recognition site is formed. This suggests that interaction between SgrAI and these two base pairs plays an important role in filamentation regulation. The mechanism by which this regulation occurs, however, is as yet unknown. In previous studies, the structure of SgrAI bound to the primary site and one type of secondary site was solved. Cryo-EM was also used to determine the filamentous structure of SgrAI bound to primary site. According to structural and kinetic studies, substitution of the second base pair of the primary site can change the energy between the inactive (T state) and active (R state) states in a way that favors the T state and, as a result, prevents the formation of filamentous structures. However, these studies did not provide any information on the substitution of the first base pair. DNA is cleaved by SgrAI using a two-metal ion mechanism. The presence of both metal ions is required for optimal cleavage rates. In the previously solved dimeric structures, only one of these atoms was found at the correct position. The other one was located about 4A away from its predicted position. The displacement of this ion in dimeric form probably contributes to the slow DNA cleavage in inactive form. A second metal remains misplaced in the filamentous structure of SgrAI with synthesized DNA that is lacking scissile phosphate. It was hypothesized that the presence of scissile phosphate would be necessary for the second metal to achieve its proper position. I used SgrAI to study filamentation mechanisms in this study. The filamentous structure of SgRAI bound to the primary site DNA containing scissile phosphate was solved to 2.5 A. In this structure, the second metal ion is located at the correct location and a conformational change is observed. It is likely that this conformational change is associated with the correct position of the second metal ion and also with the high cleavage rate. The apo structure of SgrAI has also been resolved to 2 Å. The structure revealed a disorder loop involved in the recognition of DNA. Based upon these structures, the previously proposed model of filamentation of SgrAI was remodeled into a more detailed model. In addition, the kinetic pathway of SgrAI cleavage of the primary site DNA was thoroughly investigated. However, the mechanism for cleavage of the secondary site DNA remains unclear. This study also aimed to investigate the kinetic pathway of SgrAI cleavage on secondary sites by using the same model used for the cleavage of primary sites, but by adding an additional step, an equilibrium between R and T states. Our hypothesis was that the addition of a single equilibrium between conformations with low and high activity may be able to adapt our previous computational model developed for the cleavage of primary DNA sites to the cleavage of secondary DNA sites. Experimental data indicate that binding to secondary site DNA stabilizes the T state approximately 5-17-fold over the R. In this study, the final goal was to solve the crystal structure of SgrAI bound to secondary site DNA with a single nucleotide substitution on the second base pair. The solution to this structure will allow us to confirm our proposed model for filamentation kinetics. According to our hypothesis, the R31 loop cannot form hydrogen bonds with the secondary site sequence, as it can with the primary site, so it will remain partially or completely disordered. Another DNA-binding protein on which I worked was the nonstructural protein (NS1) of human parvovirus B19. It is hypothesized that this protein possesses helicase activity in its central domain. In this study, I analyzed the isolated central domain of B19V-NS1 and investigated its helicase activity on non-specific and specific DNA sequences, ATP and ATP-g-S effects, and the state of oligomerization. Additionally, I evaluated the helicase activity using full-length NS1 and compared it to results obtained with the isolated central domain.