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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
Saccharomyces cerevisiae-based system for studying clustered DNA damages
Mario Moscariello1, Betsy Sutherland
1Brookhaven National Laboratory, Biology Department, Upton, NY 11973, USA. mario.moscariello@uk-essen.de
Radiation and Environmental Biophysics
|June 17, 2010
Summary
DNA-damaging agents can create complex lesions. Repairing these clustered DNA damages can lead to double-strand breaks (DSBs), a critical DNA damage type.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs).
- Repairing MDSs on opposing DNA strands can generate single-strand break intermediates.
- These intermediates may convert base damage into double-strand breaks (DSBs).
Purpose of the Study:
- To determine if closely opposed base damages convert to DSBs.
- To investigate the role of homologous recombination repair in this process.
- To establish a model system for studying MDS repair.
Main Methods:
- Constructed a diploid S. cerevisiae yeast strain.
- Targeted chromosomal context with integrative DNA fragments carrying clustered uracil DNA damages.
- Assessed DSB formation and homologous recombination outcomes.
Main Results:
- Closely opposed uracil DNA damages were modeled as MDSs.
- The study system demonstrated the conversion of base damage to DSBs.
- Homologous recombination repair pathway outcomes were analyzed.
Conclusions:
- The developed yeast system effectively models MDS repair.
- Closely opposed base damages can be converted to DSBs.
- This system can be extended to study various complex DNA lesions.
