Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Topoisomerase I (Top1) exhibits ribonuclease activity, creating DNA nicks with 2',3'-cyclic phosphates at ribonucleotide sites.
• Genomic ribonucleotides arise from replicative polymerases and require removal by enzymes like RNase H2.

Purpose of the Study:

• To investigate whether Topoisomerase I (Top1) can directly generate DNA double-strand breaks (DSBs) at genomic ribonucleotide sites.
• To elucidate the mechanism and consequences of Top1-induced DNA damage in the absence of RNase H2.

Main Methods:

• Genetic analysis of yeast strains with deficiencies in RNase H2, RAD52, and RAD51.
• Biochemical assays to detect Top1-induced DNA nicks and DSBs.
• Analysis of Top1-linked DNA using pull-down experiments.

Main Results:

• RNase H2-deficient yeast cells showed increased Rad52 foci, indicating DNA damage.
• Inactivation of RNase H2 and RAD52 resulted in synthetic lethality, while combined loss of RNase H2 and RAD51 caused slow growth and replication stress.
• Deletion of TOP1 rescued these phenotypes, implicating homologous recombination in repairing Top1-induced damage.
• Biochemical evidence confirmed Top1 generates irreversible DSBs at ribonucleotide sites, with Top1 covalently linked to DNA ends.

Conclusions:

• Topoisomerase I (Top1) is a direct source of DNA double-strand breaks (DSBs) when genomic ribonucleotides are not removed by RNase H2.
• This Top1 activity contributes to genome instability.
• Efficient removal of ribonucleotides by RNase H2 is crucial for preventing Top1-mediated DNA damage.