# Topoisomerase 1-catalyzed genomic ribonucleotide excision, its regulation, and its implication in transcription.

> **NIH NIH F31** · TRUSTEES OF INDIANA UNIVERSITY · 2022 · $30,752

## Abstract

PROJECT SUMMARY
In eukaryotes, ribonucleotides are frequently incorporated into DNA during replication (1 ribonucleotide per every
1000-5000 deoxyribonucleotides). Canonically, RNase H2 is the protein responsible for the removal of these
embedded ribonucleotides. However, it has been recently shown that topoisomerase 1 (Top1) also has its own
genomic ribonucleotide processing activity. When this processing occurs in specific short repeat sequences, it
can lead to 2-7 bp deletions. These deletions are the result of two sequential nicks by Top1 that releases a small
single-stranded DNA segment and is then followed by a strand slippage and ligation across the formed gap.
These deletions have been shown to be biased towards the non-transcribed strand (NTS) of highly transcribed
genes, but the reasoning for this strand specificity is yet to be elucidated. With the help of our recent preliminary
data, we propose that this strand specificity for Top1 activity is due to the formation of DNA topological structures,
specifically negative supercoils, behind the RNA polymerase that bias the initial cleavage by Top1. This would
re-define the way we think about Top1-mediated DNA relaxation by limiting the cleavage of Top1 mainly towards
the NTS during transcription. This limitation also allowed us to hypothesize about a possible biological implication
for Top1-catalyzed excision of ribonucleotides. When Top1 cleaves a ribonucleotide, it can generate a unique
nick lesion known as 2’,3’-cyclic phosphate (CP). The bias of Top1 cleavage at the NTS and the formation of
CPs lead us to hypothesize that this transient nick lesion could serve as a way of continually relieving
transcriptional torsional stress, as CPs would be forming at a strand that would potentially not interfere with
transcription, unlike nicks at the transcribing strand. We plan to investigate this by looking at mRNA expression
after depleting genomic ribonucleotides in yeast. This investigation will provide us with potential roles of
ribonucleotide incorporation in eukaryotes. Additionally, the processing of genomic ribonucleotides by Top1
produces PARP-trapping lesions, a chemotherapeutic target for BRCA1/BRCA2 deficient tumors. I will look at
the interactions that could lead to the trapping of PARP1 after ribonucleotide cleavage by Top1 and its link to the
ability of PARP1 to regulate CP formation (identified recently by us and shown in our preliminary data). The
presence of either CPs or adducts between DNA and Top1 are likely candidates for the recruitment of PARP1.
Overall, our findings will help clarify the relevance of ribonucleotide incorporation for transcriptional regulation by
providing insight into a novel mechanism of DNA relaxation by Top1 through the processing of those genomic
ribonucleotides. Our proposal will also aim to describe the regulation of CP formation by PARP1, and the
physiological relevance of the interaction between Top1, PARP1, and genomic ribonucleotides.

## Key facts

- **NIH application ID:** 10536476
- **Project number:** 1F31CA268751-01A1
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** Edward James Sarrain
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $30,752
- **Award type:** 1
- **Project period:** 2022-09-06 → 2026-09-05

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10536476

## Citation

> US National Institutes of Health, RePORTER application 10536476, Topoisomerase 1-catalyzed genomic ribonucleotide excision, its regulation, and its implication in transcription. (1F31CA268751-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10536476. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*