Monitoring DNA double-stranded break repair (DSBR) homology search in situ via RAD51 proximity labeling
EMBARGOED UNTIL 2027-12-06
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Date
2024
Publication Type
Doctoral Thesis
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yes
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EMBARGOED UNTIL 2027-12-06
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Abstract
Maintaining genome integrity is a vital function in living organisms, and accumulating uncorrected DNA damage may lead to malfunction and pathogenesis. However, DNA damage takes on a wide variety of forms and there are an equally diverse number of DNA repair pathways. Among the most toxic DNA damage events are double-stranded breaks (DSBs). In many forms of damage only one strand of DNA is affected, and this damage is readily corrected using the complementary strand as an intact donor template. In DSBs, this possibility does not exist, and it is then unknown whether mutations have occurred at the break site. A choice must then be made, find a template to restore any lost information or repair the DSB with the risk of mutations.
Whereas non-templated repair is fast, this process is often mutagenic, leading to insertion or deletion (indel) mutations. Templated repair—also called homology directed repair (HDR)—is significantly slower in human cells and requires a careful comparison of the DSB to many candidate DNA sequences to copy information from before ultimately choosing one. This process is called homology search and is a key rate limiting step during HDR. The ideal HDR donor template is often the sister chromatid, a duplicate of the genome made in preparation for cell division. However, only the small region exactly corresponding to the DSB site should be used, recombination with any other genetic sequence will cause mutagenesis. Therein lies the problem, finding a single short DNA sequence out of an enormous background, being 6.3 billion DNA bases in humans.
Despite the importance of homology search in HDR, limited methods exist to study this process. The available techniques are constrained by low-throughput, poor sensitivity, or lack of DNA sequence resolution. Thus, in this work we develop a novel method that enables highly sensitivity, in situ/in vivo, and sequence-resolved monitoring of HDR homology search. This method extends upon an earlier DNA-protein proximity labelling method called DamID. As the key protein involved in DSBR homology search is RAD51, we term our novel method RAD51 proximity identification sequencing (RaPID-seq). Using RaPID-seq, we provide new insight into homology search dynamics in human cells. In contrast to bacteria and budding yeast, we find that homology search is constrained to the region proximal to the DSB site. Furthermore, we extend the findings and method of RaPID-seq into studying the dynamics of genome editing and multiplexing with other important molecular biology techniques, demonstrating the potential of RaPID-seq as a core technique of DNA repair research.
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published
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ETH Zurich
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Subject
DNA repair; double-strand breaks; CRISPR; homology-directed repair; Next generation sequencing (NGS)
Organisational unit
09635 - Corn, Jacob / Corn, Jacob
Notes
Funding
188858 - Molecular mechanisms of human genome editing (SNF)
Related publications and datasets
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