Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Kristina Schmidt, Ph.D.

Committee Member

Mark Alexandrow, Ph.D.

Committee Member

Marcus Cooke, Ph.D.

Committee Member

Huzefa Dungrawala, Ph.D.

Keywords

RAD5, MUS81, Replication Fork Restart, POL30

Abstract

DNA replication needs to be strictly monitored to ensure proper duplication of the genome. During DNA synthesis, the DNA replication machinery encounters multiple obstacles such as incorrect dNTP incorporation, RNA-DNA hybrids, modification of DNA nucleobases, collision with the transcription-replication machinery. These endogenous and exogenous sources of DNA damage may result in single-strand breaks (SSBs) and double-strand DNA breaks (DSBs), thereby impeding fork progression. To prevent stalling of DNA synthesis a multitude of DNA repair pathways are specifically designed to deal with these kinds of blockages. One of the proteins involved in ensuring DNA replication fork progression is the DNA helicase Rrm3 in Saccharomyces cerevisiae. Rrm3 belongs to the PIF1 DNA helicase family, which is evolutionary conserved from bacteria to humans. The structure of members of the PIF1 DNA helicase family can be divided into its helicase domain located in the C-terminus and a disordered N-terminus, which promotes protein-protein interaction. Rrm3 interacts with the subunit Orc5 of the origin complex and possesses a PCNA-interacting protein-box (PIP-Box) in its N-terminus. The majority of Rrm3’s function can be attributed to its 5’-3’ DNA helicase activity as deletion of RRM3 or disrupting its Walker A motif leads to increased replication fork stalling. Therefore, yeast cells deficient of Rrm3 in S. cerevisiae were used as a model organism to study how cells deal with increased stalled replication forks. One of the proteins identified to be upregulated in the presence of increased stalled replication forks is Rad5. Rad5 and its human orthologue, HLTF, belongs into the SWI/SNF family. Rad5 possesses a conserved region encoding an ATPase domain and seven helicase-related sequence motifs in its C-terminus and a HIRAN domain in the N-terminus. Both domains are important for fork reversal of stalled replication forks under replication stress. Furthermore, a really interesting new gene (RING) ubiquitin-ligase motif is embedded within the helicase domain, allowing Rad5 in a complex with Mms2-Ubc13 to polyubiquitinated PCNA. This in turn initiates the error-free template switching pathways using the newly synthesized sister strand as template to bypass the DNA lesion. In the second chapter I identify which function of Rad5 is required to deal with increased stalled replication forks in the absence of Rrm3. We determine that the Helicase and HIRAN domain, which are both involved in fork reversal activity, suppress replication stress in the absence of Rrm3, whereas the ubiquitin-ligase activity is not. Furthermore, prolonged fork stalling in the absence of Rrm3 and Rad5 results in recombinogenic DNA lesions, which are being processed by a Rad59-dependent recombination salvage pathway. These recombinogenic DNA lesions are dependent on Rrm3’s helicase activity, but not its N-terminus and on Rad5’s fork reversal activity. However, the ubiquitin-ligase activity of Rad5 and therefore poly-ubiquitination of PCNA is dispensable. Moreover, I identify the structure-specific endonuclease Mus81 to be required to prevent recombinogenic DNA lesions and gross chromosomal rearrangements (GCRs) in the absence of Rrm3, but not Rad5. Thus, two independent mechanisms, one depending on Rad5’s fork reversal activity and the other one on Mus81 dependent cleavage, exist to bypass fork stalling at replication barriers, thereby maintaining genome stability. The third chapter focuses on further exploring the biological relevance of the interaction between Rrm3 and Pol30. I identified a positive genetic interaction between RRM3 and POL30 mutant, which is unable to modify lysine 127, which is located on the inter-domain connecting loop (IDCL). Additionally, deletion of RRM3 in other pol30 mutants, when either the ubiquitination site (pol30-K164R) or both, the SUMOylating and ubiquitination site (pol30-K127,164R) are mutated, did not reveal a genetic interaction. Deletion of RRM3 suppresses the DNA damage sensitivity and the accumulation of recombinogenic DNA lesions in the pol30-K127R mutant. Surprisingly, deletion of Rrm3’s N-terminus, specifically, the first 54 amino acids containing the PIP-box are required for the suppression of the DNA damage sensitivity and for the recombinogenic DNA lesions, but not its helicase activity. Furthermore, the suppression of pol30-K127R mutant in the absence of Rrm3 depends on an unknown substrate of the SUMO E3-ligase Siz1 and to a lesser extent Siz2. Thus, it appears that the physical interaction with PCNA, when lysine 127 cannot be SUMOylated, causes DNA damage sensitivity and accumulation of recombinogenic DNA lesions rather than Rrm3’s catalytical activity. Finally, studying the cellular functions and biochemical characteristics of Pif1 DNA helicases in yeast, such as Rrm3, can help to gain a better understanding on how the human orthologue PIF1 DNA helicase functions as tumor suppressor.

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