Dissertation
Sources and signatures of mutagenesis in break-induced replication
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005668
Abstract
Break-induced replication (BIR) is a type of double strand break (DSB) repair in which a single broken DSB end (like that resulting from an eroded telomere or collapsed replication fork) can invade into a homologous DNA template to initiate repair. BIR proceeds via a migrating D-loop, often continues for hundreds of kilobases, and results in conservative inheritance of the newly synthesized strand. Because leading and lagging strand synthesis are asynchronous during BIR, a long track of ssDNA accumulates as the D-loop migrates. Persistent ssDNA formed by BIR is highly susceptible to damage, which in turn makes it mutagenic. Additionally, BIR that is interrupted or stalled can produce genomic rearrangements or switch to microhomology mediated BIR (MMBIR). MMBIR is a DNA repair pathway initiated by polymerase template switching at microhomology, which can produce chromosomal rearrangements underlying several neurological and metabolic diseases and was proposed to promote complex genomic rearrangements (CGRs) that contribute to cancer. Yet, the extent of MMBIR activity in genomes is poorly understood due to difficulty in directly identifying MMBIR events by whole genome sequencing (WGS). In this thesis, by using our newly developed MMBSearch software, we directly detect MMBIR events in human genomes and report substantial differences in frequency and complexity of MMBIR events between normal and cancer cells. Through analysis of 71 cancer genomes from The Cancer Genome Atlas (TCGA), we observed that MMBIR mutations readily accumulate de novo across several cancer types, with particularly high accumulation in some breast and lung cancers. By contrast, MMBIR events appear only as germline variants in normal human fibroblast cells, and do not accumulate as de novo somatic mutations. Finally, we performed WGS on a lung adenocarcinoma patient case and confirmed MMBIR-initiated chromosome fusions that disrupted potential tumor suppressors and induced chromothripsis-like CGRs. Our findings newly document MMBIR as a trigger for widespread genomic instability and highlight MMBIR as a potential driver of tumor evolution.
A second goal of this thesis was to understand how ssDNA formed during BIR produces mutations when combined with DNA damage or when synthesis encounters DNA sequence motifs prone to non-B DNA structure formation. In this thesis we assess the mutagenicity of ssDNA intermediates of BIR when exposed to the cytosine deaminating enzyme APOBEC3A (A3A) and formation of hairpin structures at quasi-palindromic sequence. Using an inducible BIR system in the budding yeast Saccharomyces cerevisiae, we show that quasi-palindromic sequences placed on the track of BIR promote deletions by replication slippage over hairpin structures that form in ssDNA of both the lagging strand and within the BIR D-loop. In a similar BIR-inducible system with a reporter cassette specific to A3A-induced mutagenesis in the template for leading strand synthesis, we determined that ssDNA within the BIR D-loop is susceptible to DNA damage by A3A along the entire track of BIR. Finally, we show that damage inflicted by A3A on the template within the D-loop is not efficiently excised by the DNA-glycosylase Ung1. In total, our data newly identify ssDNA within the BIR D-loop as a potent source of mutagenesis.
Details
- Title: Subtitle
- Sources and signatures of mutagenesis in break-induced replication
- Creators
- Beth Anne Osia
- Contributors
- Anna Malkova (Advisor)Terry Braun (Committee Member)Albert Erives (Committee Member)Jan Fassler (Committee Member)Sarit Smolikove (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biology
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005668
- Publisher
- University of Iowa
- Number of pages
- xv, 176 pages
- Copyright
- Copyright 2020 Beth Anne Osia
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 154-163).
- Public Abstract (ETD)
Double-strand breaks (DSB) in DNA occur for a variety of reasons including damage from environmental factors and normal cellular processes. While these breaks must be repaired to continue normal cellular function, repair comes at a price, with some repair pathways producing harmful mutations and rearrangements as a byproduct. Here, we investigate two DSB repair mechanisms that accumulate damage and that produce mutations: - microhomology mediated break induced replication (MMBIR), and single-stranded DNA (ssDNA) accrued during break-induced replication (BIR).
The first goal of this work was to develop a new software to identify MMBIR in human genomes based on the mutational footprint we previously defined using baker’s yeast as a model. We used this software, which we named MMBSearch, to identify MMBIR mutations in cancer genomes, where they occur more often than in normal genomes. The findings of this work may lead to new targets for anti-tumor drugs that target the MMBIR pathway. The second goal of this work was to understand how specific DNA structures formed during repair by BIR, produce mutations when combined with DNA damage or DNA sequence motifs prone to secondary structure formation. We show that accumulated ssDNA and D-loop-associated ssDNA are vulnerable to secondary structure formation and DNA damage, and that both lead to increased mutagenesis during BIR. These findings provide additional insights into the mechanisms by which repair by BIR can produce mutations.
- Academic Unit
- Biology
- Record Identifier
- 9984036790802771
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