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Dissertation
Break-induced replication as a DNA double-strand break repair pathway: mutagenic consequences and mechanistic insights
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2025
DOI: 10.25820/etd.008158
Abstract
DNA, the fundamental hereditary molecule, is susceptible to damage, including double-strand breaks (DSBs). DNA DSBs need to be repaired for the cell to survive and if not repaired correctly, DSBs can lead to genomic instabilities including point mutations, insertions and deletions, genomic rearrangements, and aneuploidies. Such genomic instabilities have been implicated in aging and many human diseases such as cancer. Break-Induced Replication (BIR) is a DNA repair mechanism that repairs one-ended DNA DSBs. However, BIR is error-prone, often leading to genomic rearrangements and with nucleotide substitution error rates 1000 times higher than S-phase replication. High rate of mutagenesis during BIR has been tied to the extensive amounts of single-strand DNA forming on the BIR’s lagging strand. This ssDNA is highly susceptible to damage by mutagens or to spontaneous deamination of cytosine residues, often leading to mutations.
By utilizing an inducible BIR system in Saccharomyces cerevisiae, this dissertation demonstrates novel findings that BIR not only generates mutagenic ssDNA on its lagging strand but also on the leading strand template (D-BTS). Specifically, we show that this D-BTS ssDNA is susceptible to difficult-to-repair damage at cytosines. We also show that D-BTS ssDNA can form hairpin-like secondary structures leading to deletions.
Furthermore, our research demonstrates that during meiosis—the specialized cell division creating egg and sperm cells—both BIR and another DNA repair process called DNA end resection can generate long tracts of mutagenic ssDNA in a DSB-dependent manner. Here, by meiotically expressing human cytosine deaminase APOBEC3A (A3A) which specifically attacks ssDNA, we provide the first experimental evidence explaining how ssDNA mutagens can promote mutation clustering during meiosis, potentially contributing to infertility and congenital diseases. Moreover, we demonstrate that short tracts of ssDNA can form within gene promoter regions during meiosis independently from DNA DSBs. Finally, we provide the first evidence suggesting that during meiotic recombination single nucleotide polymorphisms (SNPs) between parental DNA sequences can lead to heteroduplex rejection by Msh2 which in turn leads to more extensive amounts of mutagenic ssDNA in meiosis.
Additionally, this research investigated a related and poorly understood error-prone DNA repair pathway called Microhomology-Mediated BIR (MMBIR). MMBIR has been proposed to utilize short regions of microhomology to promote polymerase template switching leading to templated insertions and rearrangements that underly neurological diseases and cancer. However, due to computational challenges in mapping whole genome sequencing (WGS) reads containing MMBIR mutations, the prevalence and the functional impact of MMBIR in both cancer and in healthy human populations is still not well understood. In this work, we report the frequency of MMBIR mutations within exomes from healthy human populations within the 1000 Genomes Project as well as across twelve cancer studies within The Cancer Genome Atlas (TCGA). We show that certain cancers have elevated genic MMBIR burden as compared to healthy human populations. We also show that high MMBIR burden can be associated with differing levels of somatic mutation burden and with complex changes in gene expression, depending on the type of cancer.
Altogether, this work advances our understanding into how DNA DSB repair processes during meiosis and in cancer lead to formation of mutagenic ssDNA which can lead to mutations and mutation clusters driving disease, cancer, and evolution.
Details
- Title: Subtitle
- Break-induced replication as a DNA double-strand break repair pathway: mutagenic consequences and mechanistic insights
- Creators
- Jerzy Mateusz Twarowski
- Contributors
- Anna Malkova (Advisor)Josep M Comeron (Advisor)Bin He (Committee Member)Veena Prahlad (Committee Member)Natalie Saini (Committee Member)Jan Fassler (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biology
- Date degree season
- Summer 2025
- DOI
- 10.25820/etd.008158
- Publisher
- University of Iowa
- Number of pages
- xx, 285 pages
- Copyright
- Copyright 2025 Jerzy Mateusz Twarowski
- Language
- English
- Date submitted
- 07/28/2025
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 260-277).
- Public Abstract (ETD)
DNA, the blueprint for life, is susceptible to damage, including double-strand breaks (DSBs). If not repaired correctly, these DSBs can lead to harmful mutations, contributing to aging and diseases including cancer. This dissertation investigates the mechanisms by which the error-prone DNA repair pathway called Break-Induced Replication (BIR) contributes to mutagenesis and genomic instability.
This dissertation presents novel findings, including the first evidence that BIR generates tracts of single-stranded DNA (ssDNA), a highly mutagenic form of DNA, on its leading strand DNA template. Specifically, this template ssDNA is shown to be highly susceptible to damage and to deletions at sequences forming secondary structures. Furthermore, this research demonstrates that during meiosis the specialized cell division creating egg and sperm cells both BIR and another DNA repair process called DNA end resection can generate long tracts of mutagenic ssDNA.
This thesis also provides the first experimental evidence explaining how ssDNA mutagens can promote meiotic mutation clustering, potentially contributing to infertility and congenital diseases. Additionally, this research investigated a related and poorly understood error-prone DNA repair pathway called Microhomology-Mediated BIR (MMBIR). Specifically, mutational patterns characteristic to MMBIR were identified within various cancers, pointing to MMBIR's potential role in development of certain cancers.
Collectively, this work advances our understanding into how DNA repair processes during meiosis and in cancer lead to mutations that drive disease, cancer, and evolution.
- Academic Unit
- Biology
- Record Identifier
- 9984948238102771
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