All ETDs from UAB

Advisory Committee Chair

Mary-Ann Bjornsti

Advisory Committee Members

David Schneider

John M Parant

Eddy S Yang

Bradley K Yoder

Document Type

Dissertation

Date of Award

2018

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

DNA replication is a highly conserved and tightly regulated process, with a variety of factors acting to ensure faithful duplication and stability of chromosomal DNA. During replication, positive supercoils form ahead of the advancing replication fork. DNA topoisomerase 1 (Top1) acts to prevent DNA damage by relieving the torsional strain created by these supercoils. The camptothecin (CPT) class of chemotherapeutics target Top1, creating a long-lived DNA-Top1-CPT covalent complex, which leads to S-phase specific DNA lesions and cellular death. Our lab previously performed a genetic screen to isolate mutant proteins that enhance cell sensitivity to Top1-mediated DNA damage. One of the mutant proteins identified was Cdc45G510E, which contains a single amino substitution at residue G510. Cells expressing this protein were also hypersensitive to other forms of DNA damage and exhibited collapsed replication forks, suggesting a role for Cdc45 in protecting cells from genotoxic stress. Cdc45 is an essential protein found within the CMG (Cdc45-MCM-GINS) replicative helicase. Until recently, it was considered a scaffolding protein that had only been identified in eukaryotes. However, sequence alignments and structural studies suggest that Cdc45 may in fact be the eukaryotic ortholog of the archaeal RecJ protein. This is due, in large part, to the conservation of residues within DHH (Asp-His-His) motifs in the N-terminus of Cdc45, which act to coordinate a metal ion and are essential for the nuclease activity of various RecJ proteins. The functional similarities between Cdc45 and RecJ identified thus far are the inclusion with the respective replicative helicases and the ability to bind ssDNA. RecJ has also been determined to aid in DNA damage repair, such as homologous recombination and rescue of stalled replication forks. In our mutational analyses of yeast CDC45, we demonstrated that the conserved DHH residues were also necessary for Cdc45 function in protecting cells from genotoxic stress. Moreover, several of these residues are also required to maintain cell viability in the absence of exogenous DNA damage. Through the studies described in this dissertation, we have provided two models for Cdc45 function which predict that the distinct defects seen with Cdc45G510E and the Cdc45-DHH mutations would elicit similar phenotypes of enhanced sensitivity to replicative stress, accumulation of ssDNA and activation of the S-phase checkpoint in the absence of replicative stress, and replication fork instability.

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