Dissertation
Investigation of meiotic double-strand break formation through epitope-tagged SPO-11 and the study of its novel regulator, and the recruitment of early repair proteins as function of age and sex of C. elegans
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2023
DOI: 10.25820/etd.007077
Abstract
Meiosis is a cell division in which an organism prepares for sexual reproduction by producing gametes. To successfully complete meiosis, meiotic cells undergo homologous recombination (HR) between paired homologous chromosomes. Recombination begins with the induction of programmed double-strand breaks (DSBs) and is concluded with the formation of crossover or non-crossover events. Here we aimed at better understanding of how aging effects the repair of DNA damage by monitoring how the conserved RPA complex responds to induced DSBs in young and old worms. We also utilized methods to induce both simple and complex DNA damage in males to gain better understanding of potential differences in DNA damage response between male and hermaphrodite C. elegans. We identified the previously uncharacterized protein EEED8.3 as a repressor of meiotic DSBs. Finally, we used the conserved topoisomerase/TOP2-like protein SPO-11 to gain better understanding of the formation of programmed DSBs by modifying SPO-11 with an epitope tag, allowing us to directly observe the activity of SPO-11 for the first time in metazoans.
In response to DNA damage, the dsDNA at the site of breakage is resected by the MRN-C complex (MRE-11, RAD-50, NBS-1, and COM-1). The ssDNA is bound by the RPA complex and then replaced by RAD-51 to protect the DNA from degradation and facilitate homolog invasion and repair. Modification of the histone H3K9me2 by HIM-17 promotes DSB formation by SPO-11, which covalently binds to DNA and is removed by the MRN-C complex. In the absence of MRN-C, SPO-11-bound to sites that do not become DSBs creates DNA-protein crosslinks (DPCs) which require alternative removal methods. In metazoan germ cells, DPCs formed by similar proteins, named TOP2, are repaired by the metalloprotease GCNA via SUMOylation by the E2 conjugating enzyme Ubc9, resolving TOP2-DPCs via protein digestion followed by the excision of remaining fragments by the activity of TDPT2. The HR and DPC resolution proteins are found in different DNA damage response pathways in the hermaphrodite germline. There are also different DNA damage responses in male C. elegans from hermaphrodites. These differences include the lack of apoptotic removal of damaged germ cells, and induced damage resulting in increased fertility. The causes of these differences are part of the aim of this thesis.
In this thesis I investigated four different topics, all in the aim of understanding DSB repair in the germline of the nematode C. elegans. In the first, we utilized SPO-11 with an epitope tag to gain direct insight into the activity of SPO-11 in meiosis. We find evidence of previously unknown effects of meiotic and repair proteins on the efficacy of SPO-11-chromatin association and DSB formation. We report the requirement for association of HIM-17 and DSB-1 to chromatin, priming SPO-11 DSB sites during mitotic proliferation to allow rapid SPO-11-chromatin association upon meiotic entry. Unlike wild type SPO-11, we find evidence that SPO-11::HA forms DPCs that can be resolved by GCNA-1 and TDPT-1 for proteolysis and excision. The presence of these proteins prevents the activity of alternate DPC removal proteins. These discoveries uncover evidence of a heretofore unknown alternate repair pathway to remove SPO-11 DPCs. Our studies indicate that the ability to directly visualize SPO-11 activity provides unprecedented insight into its activity and regulation. In the second project we identify the previously uncharacterized protein EEED8.3 (hereafter RDF-1) as a suppressor of DSB formation. We find that the double mutant increases the number of RAD-51 foci during pachytene, indicating a role in the suppression of DSB formation. Therefore, the discovery of rdf-1 allows additional detail to be added to the current model of formation of programmed DSBs. In the third, we utilize the localization of MRE-11 and EXO-1 to investigate male damage repair. Observing the recruitment of MRE-11 and EXO-1 paired with GFP in response to complex and simple exogenous damage allowed us to compare differential responses in male and hermaphrodite C. elegans, though further study to fully elucidate these differences is required. Finally, we investigated the effect of aging on meiosis via the activity of the RPA complex in response to exogenous DNA damage. We found that expression of RPA-1 does not change as the worm ages. However, both recruitment to and removal of RPA from induced DNA damage is impaired by age. The research presented in this thesis provides insight into meiotic DNA damage repair and the first direct observation of how meiotic DSB repair proteins influence the behavior of programmed DSBs.
Details
- Title: Subtitle
- Investigation of meiotic double-strand break formation through epitope-tagged SPO-11 and the study of its novel regulator, and the recruitment of early repair proteins as function of age and sex of C. elegans
- Creators
- Richard Bowman
- Contributors
- Sarit Smolikove (Advisor)Bryan Phillips (Committee Member)Anna Malkova (Committee Member)Marc Wold (Committee Member)John Logsdon (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Summer 2023
- DOI
- 10.25820/etd.007077
- Publisher
- University of Iowa
- Number of pages
- xvii, 187 pages
- Copyright
- Copyright 2023 Richard Bowman
- Language
- English
- Date submitted
- 07/21/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 168-187).
- Public Abstract (ETD)
Meiosis is the process of preparing for reproduction through the creation of gametes. Gamete production requires successfully completing homologous recombination in order to transmit a one of each parental chromosome into the germ cell. This process begins with the induction of programmed double-strand breaks (DSBs), initiating the process that is leading to a crossover connecting homologous chromosomes, insuring proper segregation at the completion of meiosis I and II. There are many unanswered questions regarding how DNA damage is repaired in meiosis, including: the effects of aging on meiotic DNA damage repair, how differently meiotic damage is repaired in different sexes, and how to study directly the activity of proteins directly involved in programmed DSBs.
Here, we show that aging detrimentally effects the ability to repair DNA damage in germ cells. We have identified a novel gene involved in the regulation of meiotic DSBs, and we have found evidence of differential repair between male and hermaphrodite C. elegans. The protein that initiates programmed DSBs, Spo11, is highly conserved and as such the ability to directly visualize its activity would shed light on the entire process. We have successfully integrated an epitope tag into an internal location of the C. elegans SPO-11 protein that has allowed us to directly visualize its localization to chromatin throughout the germline. By utilizing tagged SPO-11 we have found that the endonuclease function of MRE-11 is critical for DSB formation and crossover. Our data suggests that the protein HIM-17 and the RMM complex are required for priming chromatin for SPO-11::HA localization on meiotic entry, and that SPO-11::HA DNA-protein crosslinks are processed by GCNA-1, TDPT-1, and UBC-9. Additionally, we identified a novel suppressor of DSB formation, as well as characterizing how aging impacts DSB repair in aging hermaphrodites. Our analysis provides an increase in the understanding of meiotic programmed DSB formation and repair. As many of the genes involved in this study are highly conserved, our findings can be applied to the study of other model organisms.
- Academic Unit
- Biology
- Record Identifier
- 9984454742302771
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