Dissertations, Theses, and Capstone Projects

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

David Jeruzalmi

Committee Members

Anuradha Janakiraman

Stefan Pukatzki

Amedee des Georges

Michael O’Donnell

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Keywords

Nucleotide Excision Repair, DNA replication initiation, Bacterial DNA repair and replication, multipartite genome, UvrA-DNA, RctB-oriCII

Abstract

Nucleotide excision repair (NER) is one of the fundamental DNA repair pathways in all organisms. A unique facet of NER is the large set of DNA lesions the pathway is capable of repairing (Truglio, Croteau, et al. 2006). The NER proteins may thus be responding to global features of native DNA such as changes in thermodynamic stability, geometry and kinetic properties (Maillard et al. 2007). In this study we set out to investigate the first and primary responder to NER in bacteria, UvrA (Kraithong et al. 2021). UvrA is a homo-dimeric ABC ATPase; proteins that are known to transduce the information from ATP binding and hydrolysis to function (Thomas and Tampé 2020). Our goal was to gain a deeper understanding of UvrA’s ability to distinguish DNA lesions displaying different chemical and geometric properties. To this end we utilized cryogenic electron microscopy (cryo-EM) for a structural understanding of UvrA with DNAs containing either a large, bulky adduct (fluorescein) or DNA lacking 3 nucleotides. Questions centered around molecular mechanisms require high spatial resolution, at least at the level of individual residues. Typically, one or more detailed structures providing insights on how the general shape and atomic features of the protein gives rise to its function. Cryo-EM is capable of offering: a near native representation of macromolecules and the ability to derive multiple high-resolution structures from one preparation.

Our analysis, conducted in the presence of ATPgS or ATP, has elucidated intricate details of the reaction mechanism as UvrA engages with damaged DNAs. Specifically, we unveil a novel dimeric arrangement of UvrA, with all four nucleotide binding sites fully engaging the bound ATPgS. This prompts an unwinding of the DNA’s central segment. The rotational movement between monomers transmits torque to the center of the bound DNA, with the polymer ends being clasped by the Sig-II domains. Subsequently, the distal nucleotide binding site opens shifting the Sig-I domain. The attached DNA-binding insertion domain draws nearer to the lesion, while the UvrB binding domain takes on a state poised for UvrB loading.

We captured an intermediate state wherein the distal site of one of the Sig-I domains is partially opened or ajar. Opening of this site restores symmetry across the dimer and places both insertion domains in proximity to the lesion. Next, we observe that in the ADP bound state the NBD-II unit rotates towards NBD-I in each monomer. This displacement happens sequentially, where the ATP-II domain of one monomer first rotates towards its distal binding site partner. Following this rearrangement of the NBD-II module, the DNA is further distorted and contacts by the Sig-II domain are shifted closer to the middle of the DNA.

Moreover, our analysis revealed a solution structure of apo UvrA and a tetrameric assembly. In this oligomeric state the DNA binding groove is obstructed by the insertion domains of the opposing dimer and may thus represent an inhibitory state.

DNA replication ensures the propagation and transmission of biological information from parents to progeny. The initiation of DNA replication regulates this information flow and must be stringently controlled to prevent polyploidy or information loss to offspring. Initiation is contingent upon the coordination of the initiator and the replicator. In bacteria, the initiator is the evolutionarily conserved DnaA AAA+ ATPase and the replicator is a specific locus on DNA from which replication begins, called the origin of replication (oriC)(Hansen and Atlung 2018). In V.cholerae the secondary chromosome utilizes a specific initiator, RctB, in conjunction with a replicator, oriCII (Egan and Waldor 2003). RctB recognizes particular sequences within oriCII, referred to as 12-mers, which are highly conserved and indispensable for the viability of the Vibrio species (Egan and Waldor 2003). However, unlike the well-characterized DnaA-oriC system in E.coli, there exists a paucity of information concerning the interactions between RctB and oriCII.

Through a combination of biochemical and biophysical analysis we demonstrate the following: 1) definitive evidence of RctB binding to DNA sequences extending beyond the 12-mers, 2) the observation of RctB binding with varying affinities to the iterons present in oriCII, 3) binding of RctB to iteron DNA occurs in the monomeric form of the initiator, 4) elucidation of the role of the C-terminal domain in enhancing DNA binding to multivalent sites through protein-protein interactions, and 5) identification of the inhibitory effect of the C-terminal domain when RctB binds to single sites within oriCII. Notably, RctB exhibits behavior that combines features of both the DnaA-oriC and plasmid initiator-ori systems. OriC harbors distinct DnaA boxes, encompassing both strong and weak motfis, a characteristic mirrored by the iterons within oriCII-min. Additionally, plasmid initiators in their dimeric configuration typically lack replication competence, akin to RctB, which initiates DNA replication in its monomeric form.

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