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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Published on: September 11, 2022
Interaction of Saccharomyces cerevisiae HMO2 domains with distorted DNA
1Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, United States.
Abstract:
The Saccharomyces cerevisiae high mobility group protein HMO2 is a component of the chromatin remodeling complex INO80. In this capacity, it has been shown to direct INO80 to DNA double-strand breaks, thereby contributing to double-strand break repair. Consistent with such function, HMO2 binds DNA ends, protecting them from exonucleolytic degradation. We show here that both domains of HMO2, HMO2-BoxA and HMO2-BoxB, bind preferentially to distorted DNA, with HMO2-BoxA binding preferentially to four-way DNA junctions and DNA with tandem mismatches and HMO2-BoxB binding four-way junctions as well as DNA with stem-loop structures, tandem mismatches, and abasic sites. As previously reported for mammalian high mobility group proteins, the acidic C-terminal extension significantly attenuates DNA binding. Notably, the unique ability of HMO2 to protect DNA ends is conferred by the Box A domain. Considering the reported roles for INO80 in other events such as recovery of stalled replication forks and nucleotide excision repair, we assessed the effect of DNA damaging agents on an hmo2Δ strain; while modest growth inhibition is seen upon exposure to UV light, exposure to hydroxyurea, which causes replication fork arrest, induces severe growth deficiency. These data suggest that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks; the preferred binding of HMO2 domains to damaged DNA and intermediates in homologous recombination is consistent with such function.
Insights
The Saccharomyces cerevisiae high mobility group protein HMO2 binds distorted DNA and protects DNA ends. HMO2 is crucial for repairing DNA damage and cell survival, particularly when replication forks stall.
Area of Science:
- Molecular Biology
- Chromatin Biology
- DNA Repair Mechanisms
Background:
- The Saccharomyces cerevisiae high mobility group (HMG) protein HMO2 is part of the INO80 chromatin remodeling complex.
- HMO2 has been implicated in directing the INO80 complex to DNA double-strand breaks, aiding in their repair.
- HMO2 binds DNA ends, offering protection against exonucleolytic degradation.
Purpose of the Study:
- To investigate the DNA binding preferences of HMO2 domains.
- To determine the role of HMO2 in cellular response to DNA damaging agents.
- To elucidate the functional significance of HMO2 in DNA repair and replication fork stability.
Main Methods:
- Analysis of DNA binding specificities for HMO2 domains (HMO2-BoxA and HMO2-BoxB).
- Assessment of the impact of DNA damaging agents (UV light, hydroxyurea) on an hmo2Δ yeast strain.
- Evaluation of growth inhibition and cellular defects in response to genotoxic stress.
Main Results:
- Both HMO2-BoxA and HMO2-BoxB domains preferentially bind distorted DNA structures, including four-way junctions, tandem mismatches, stem-loop structures, and abasic sites.
- The Box A domain is responsible for the protective binding of DNA ends.
- An hmo2Δ strain exhibits severe growth deficiency upon exposure to hydroxyurea, indicating a critical role in replication fork stability, with modest sensitivity to UV light.
Conclusions:
- HMO2 possesses distinct DNA-binding domains with preferences for damaged DNA structures and intermediates.
- The Box A domain of HMO2 is essential for protecting DNA ends.
- HMO2 plays a significant role in maintaining genomic integrity, particularly in response to replication stress, suggesting its involvement in directing the INO80 complex to stalled replication forks.
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