Date of Award

1-1-2018

Embargo Period

1-1-2020

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Deepak Bastia

Second Advisor

David Long

Third Advisor

Bidyut K. Mohanty

Fourth Advisor

David Kurtz

Abstract

DNA replication fork blocking protein Fob1 blocks replication fork progression unidirectionally by binding to the terminator (Ter) site in nontranscribed spacer region 1 (NTS1) in ribosomal DNA (rDNA) of S. cerevisiae. It is involved in other cellular functions, including inter-rDNA repeat recombination, rDNA silencing via recruitment of the Sir2 histone deacetylase, cell replicative lifespan, maintaining genome integrity, preventing collision between replication and transcription, and controlling rDNA repeat expansion and contraction. It is not known whether binding of Fob1 to rDNA is sufficient to cause replication fork arrest, or if a specific interaction between Fob1 and other replication proteins is also necessary for replication fork arrest. We found through yeast two-hybrid assays and through Enzyme-Linked ImmunoSorbent Assay (ELISA) that Fob1 interacts with Mcm2-7, the catalytic core of CMG (Cdc45-Mcm-GINS), the replicative helicase in S. cerevisiae. Mcm2-7 consists of 6 subunits (numbered 2 through 7), and Fob1 showed strongest interaction with the ATPase subunits Mcm6 and Mcm7. We disrupted this interaction by generating mutants of FOB1 defective in MCM6 or MCM7 interaction via low-fidelity PCR, and screening for these non-interactions via reverse two-hybrid method. DNA binding was confirmed via colony sectoring assays. These mutants were screened for retention of interaction with Fob1 itself, Rad52, and Net1, a protein used to recruit the histone deacetylase (HDAC) Sir2. We then generated knock-in strains of these mutants using DNA cassette mediated knock-ins, and performed recombination assays and two-dimensional (2D) agarose gels to assess these mutants’ effects on replication fork arrest. 2D gel data revealed that the fob1 single-point mutants did not diminish replication fork arrest. Effects of these mutants on recombination in rDNA, which requires Fob1 dimerization and fork arrest, was tested using a plasmid integration assays. Recombination assays showed that fob1 mutants showed levels of recombination comparable to levels in the wild-type. Future studies will address other possible functions of the Mcm-Fob1 interaction. Follow-up studies could look into the same functions as this study, but with stricter fob1 mutant selection. Screening of Fob1 mutants for Rad52 interaction also revealed specific interaction between Fob1 and Rad52 that may, in future studies, clarify the function of Fob1-Rad52 interaction.

Rights

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