Dissertation
Unraveling the mechanism of BIR: from genes to genome instability
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2023
DOI: 10.25820/etd.007015
Abstract
Break-induced replication (BIR) is a pathway that repairs one-ended double strand break (DSB) resulting from replication fork collapse and telomere erosion. BIR involves invading a homologous template and initiating repair synthesis through extensive displacement loop (D-loop) migration, which can proceed to the end of the chromosome. As a result, BIR is associated with conservative inheritance of the newly synthesized strand. The process of BIR is highly mutagenic that frequently generates genome-destabilizing events such as mutations and chromosomal rearrangements, ultimately resulting in genomic instability.
Pif1 is a multifunctional DNA helicase that plays various roles in maintaining nuclear and mitochondrial genomes. While Pif1 is not essential for normal DNA replication, it is crucial for long-range BIR synthesis. In the absence of Pif1, BIR is frequently interrupted and results in chromosomal rearrangements. Yet, the functional domains of Pif1 are still poorly understood. In this thesis, we used deletion analysis to identify two important functional regions of Pif1. First, we identified a nuclear localization signal (NLS) sequence, 781KKRK784, located at the carboxyl terminus of Pif1. A mutant allele of PIF1 (pif1-NLSΔ) had wild-type levels of mitochondrial function but showed defects in nuclear functions, including telomere maintenance, Okazaki fragment processing, BIR, and binding to nuclear target sites. By fusing the NLS from the simian virus 40 (SV40) T-antigen to the Pif1-NLSΔ protein, we reduced the nuclear defects of pif1-NLSΔ cells, indicating that the identified NLS region is a core functional NLS. Second, we identified five serine residues (S41, S42, S62, S70, and S72) targeted for phosphorylation at the amino terminus of Pif1. Substituting these serine residues with either alanine (a phosphorylation null mutant) or aspartic acid (phosphomimic mutant), we observed that direct phosphorylation of these five serine residues is required for various Pif1 functions, including telomere maintenance, suppression of dna2Δ lethality and BIR.
Furthermore, our lab has recently identified spindle-assembly checkpoint (SAC) genes (BUB1, BUB3 and MAD2) as new drivers of BIR. BIR is an unusual type of DNA repair that requires a complex and time-consuming process, which often takes longer than 10 hours to complete. Therefore, the involvement of the checkpoint is prominent during BIR to delay cell division until the repair is completed. In this thesis, we assessed the role of SAC genes in the process of BIR, from initiation to completion. We observed that SAC proteins are necessary for efficient BIR completion. Moreover, BUB1 and BUB3, but not MAD2, play an important role in initiation of BIR synthesis, suggesting that BUB1 and BUB3 play a previously unknown Mad2-independent role in BIR synthesis. Additionally, we found that SAC proteins not only prolong the duration of G2/M arrest but also establish an initial checkpoint arrest in the absence of DNA damage checkpoint (DDC), a major cell cycle checkpoint activated in response to DNA damage. Finally, our data suggests that a significant crosstalk exists between SAC and DDC in the maintenance of cell-cycle arrest during BIR.
The last goal of this thesis was to detect the occurrence of microhomology-mediated BIR (MMBIR) mutations in human cells. Unlike canonical BIR, which requires a long homology template, MMBIR utilizes short homology sequences, leading to genomic instability. Recently, a high level of MMBIR is often observed in human diseases including cancer, suggesting that MMBIR is a potential contributing factor to genomic instability. However, it was unclear whether MMBIR events can be reproduced and what conditions promote the formation of MMBIR events in human cells. In this thesis, we hypothesized that MMBIR may serve as an alternative DNA repair pathway triggered in error-free homologous recombination (HR)-impaired cells. By using BRCA2 (a major HR protein)-deficient cells and performing whole-exome sequencing, we successfully detected de-novo MMBIR mutations and classified four distinct types of MMBIR outcomes in human cells under laboratory settings. In conclusion, our findings demonstrate valuable experimental conditions and analytic approaches to advance our understanding of MMBIR in the future.
Details
- Title: Subtitle
- Unraveling the mechanism of BIR: from genes to genome instability
- Creators
- Rosemary Soeun Lee
- Contributors
- Anna Malkova (Advisor)Sarit Smolikove (Committee Member)Bryan Phillips (Committee Member)Christopher Stipp (Committee Member)Maria Spies (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Autumn 2023
- DOI
- 10.25820/etd.007015
- Publisher
- University of Iowa
- Number of pages
- xxi, 203 pages
- Copyright
- Copyright 2023 Rosemary Soeun Lee
- Language
- English
- Date submitted
- 08/07/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 190-203).
- Public Abstract (ETD)
In daily life, cells experience spontaneous DNA damage that must be efficiently repaired for normal cell functions. Among various types of DNA damage, double-strand breaks (DSBs) are particularly harmful. Break-induced replication (BIR) is a pathway that repairs broken DNA strands resulting from issues during DNA replication and telomere erosion. However, BIR often introduces instability in the genome, presenting challenges for maintaining overall genetic stability.
The first goal of this work was to identify important regions of the Pif1 DNA helicase protein, which is crucial for BIR. First, we identified Pif1 region responsible for the signal that guides Pif1 protein to the nucleus. Mutations in this signal region led to defects in BIR and DNA maintenance. Second, we identified small regions of Pif1 that are targeted for specific chemical modifications of the Pif1 protein that are essential for BIR to occur efficiently.
Furthermore, we recently uncovered the roles of specific genes (BUB1, BUB3 and MAD2) during BIR. BIR is a complex and time-consuming process that may take more than 10 hours to complete. These genes regulate the cell division process to ensure BIR is successfully completed before cell division. Our study described the important involvement of these genes for successful BIR completion. Surprisingly, our observations uncovered new roles of these genes required for DNA synthesis during BIR. Additionally, we found these genes not only delay cell division but also collaborate with other checkpoint proteins to maintain genome stability in response to DNA damage.
The last goal of this study was to investigate microhomology-mediated BIR (MMBIR), a type of DNA repair that is often promoted by interrupted BIR, in human cells. MMBIR is frequently observed in human diseases, including cancer. However, the process of MMBIR was not well understood. We conducted experiments using human cells lacking the BRCA2 gene, which is involved in error-free DNA repair and observed evidence of the new MMBIR mutations that occurred in laboratory conditions in human cells. This represents the first detection of MMBIR occurrence under laboratory conditions in human cells, and we were able to categorize these new MMBIR events into four distinct types. Therefore, our study suggests a valuable approach that can be utilized for future MMBIR studies.
- Academic Unit
- Biology
- Record Identifier
- 9984546849702771
Metrics
2 File views/ downloads
60 Record Views