Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

David Jeruzalmi

Committee Members

Reza Khayat

Kevin Gardner

Brian Chait

Michael O'Donnell

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Keywords

DciA, helicase loading, DNA replication initiation

Abstract

DNA replication initiation is mediated by the binding of an initiator protein to genomic origins of replication to begin the process of loading replicative helicases onto the genome. The loading of the helicase requires the action of an accessory protein, named a “loader”. Studies on well-characterized loaders have outlined how the bacterial DnaB replicative helicase is loaded via mechanisms of “Ring-Breaking” by E. coli DnaC (EcDnaC)/protein P from bacteriophage lambda (LP) and “Ring-Making” by B. subtilis DnaI. Mounting evidence points to a recently discovered candidate loader, named DciA, that shows a more widespread distribution throughout bacteria than DnaC and DnaI combined.

This dissertation describes biochemical and biophysical characterization of DciA from V. cholerae and shows that DciA proteins functions as a loader by exhibiting four characteristics found in the DnaC/LP/DnaI: 1) DciA directly binds DnaB, 2) DciA suppresses the ATPase activity of DnaB, 3) DciA enhances the ability of the DnaB to bind ssDNA, 4) DciA aids DnaB in melting a DNA fork in an ATP-dependent manner. We also find aspects that distinguish DciA from DnaC/lP/DnaI including a 6DnaB:1DciA stoichiometry that is active in loading, and a marked ability of DciA to disassemble a pre-formed DnaB hexamer. On account of DciA’s domain similarities to Ring-Breaking loaders, we propose DciA to load DnaB via Ring-Breaking and incorporate DciA’s unique characteristics into a model that is distinct from EcDnaC/LP.

Clinical studies have implicated antibodies that react against double stranded DNA (dsDNA) as being a disease driver for Systematic Lupus Erythematosus (SLE). Studies in mice show that immune responses against the Epstein Barr virus Nuclear Antigen 1 (EBNA-1) protein can produce antibodies that also react with dsDNA. Furthermore, exogenously administering such antibodies to mice can reproduce nephritis reminiscent to that seen in SLE patients. A structural basis for how these antibodies can bind to either EBNA-1 or dsDNA would greatly clarify their role in driving SLE. Such a structural understanding would necessitate comparing two structures: an antibody bound to EBNA-1 and the same antibody bound to dsDNA.

In prior work, the 3D4 monoclonal antibody was isolated from mice infected with Epstein Barr Virus. 3D4 shows in vitro reactivity against a C-terminal fragment of EBNA-1 (named LS9B) and dsDNA. This dissertation describes the crystal structure of 3D4 bound to LS9B. LS9B is observed to bind complementarity determining region loops of 3D4 and engages 3D4 using a structural epitope comprised of two discontinuous a-helices. The 3D4-LS9B interface is mediated by hydrophobic packing, hydrogen bonding, a salt bridge, and water molecules that bridge the two proteins. Bonding between 3D4 residues Asn35/Lys55 and LS9B residues Asp577/Asp581 is highlighted as a potential charge mimic of the 3D4-dsDNA interaction. A complete understanding of how 3D4 cross reacts with DNA awaits the determination of a future 3D4-dsDNA structure.

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