# Processing of lesions into DNA repair and checkpoint pathways

> **NIH NIH R01** · ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI · 2020 · $347,874

## Abstract

DNA damage comes in many forms that originate from intrinsic and extrinsic sources. These lesions can induce
the mutations and genome rearrangements that lead to cancer, aging and degenerative diseases. Particularly
pathological lesions are the DNA double stranded DNA breaks (DSBs), as well as collapsed replication forks
caused by barriers to replication, that can themselves promote DSB formation. To activate cell cycle checkpoints,
mechanisms that allow the time to repair these lesions, ssDNA is generated at these sites in a 5’à3’ direction.
The resulting ssDNA that remains has an exposed 3’-OH group, and acts as a landing pad for assembly of
checkpoint signaling complexes as well as recombination enzymes that promote invasion into the sister
chromatid.
 Using the fission yeast Schizosaccharomyces pombe as a gene and pathway discovery tool, we identified
a family of XPG-related nucleases (XRNs) as the long sought after enzymes that are necessary and sufficient
for end resection at DSBs. This consists of the long known Rad2/Fen1 and Exo1 enzymes that also function in
Okazaki Fragment maturation and various Excision Repair pathways. The newly identified third member of this
family is known as the Asteroid nucleases. These include Ast1 in S. pombe and ASTE1 in humans, but there is
no Asteroid homolog in the budding yeast Saccharomyces cerevisiae. Thus, Ast1 enzymes remain poorly
characterized compared to Fen1 and Exo1.
 Studies leading to, and during the initial funding period of this grant, have shown that these XRN
nucleases are hierarchically recruited to DSBs, which is dynamic depending on the complement of nucleases.
There is further specificity afforded by the direction of transcription at a damaged locus. A considerable body of
data also shows that the XRNs are critical at collapsed replication forks, cooperating with several other enzymes
that modulate fork stability and processing. Additional experiments in this proposal build on these observations
and utilize an armory of new tools to study the processing of these lesions across the genome. We present a
thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its
long-studied cousins, then determine specificity determinants among them. As these initiating events in DNA
damage responses feed into many downstream response pathways, this work has significant impact on the
study of the many mechanisms that ensure the integrity of the genome.

## Key facts

- **NIH application ID:** 9972005
- **Project number:** 2R01GM124079-03
- **Recipient organization:** ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
- **Principal Investigator:** MATTHEW J O'CONNELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $347,874
- **Award type:** 2
- **Project period:** 2017-09-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9972005

## Citation

> US National Institutes of Health, RePORTER application 9972005, Processing of lesions into DNA repair and checkpoint pathways (2R01GM124079-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9972005. Licensed CC0.

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