# DNA ligase activities during base excision repair coordination

> **NIH NIH R35** · UNIVERSITY OF FLORIDA · 2023 · $200,000

## Abstract

PROJECT SUMMARY
Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of
DNA damage. BER is a multi-step pathway that requires a tight coordination between the repair proteins. The
downstream steps of BER pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing
by DNA ligase (LIGI and LIGIIIα). This step-to-step coordination is orchestrated by non-enzymatic scaffolding
protein X-Ray Repair Cross Complementing 1 (XRCC1) that plays a key role in assembling repair proteins.
Although the roles of the individual enzymes are largely studied, how the multi-protein BER complex coordinates
while maintaining the repair efficiency remains unclear. Though often considered an accurate process, the BER
can contribute to genome instability if normal coordination breaks down. Failure in the BER pathway coordination
could result in the formation of strand-break repair intermediates that are more mutagenic or toxic than the initial
DNA lesions. We hypothesize that inaccurate BER pathway coordination during DNA ligase activities
within the multi-protein complex(es) at the downstream steps of the repair response results in genomic
instability and cytotoxicity. Our parent proposal will provide the first biochemical and structural
characterization of the BER pathway coordination. We will address the main hypothesis with the following
projects: In Project 1, using biochemical and biophysical approach, we will define the molecular mechanism by
which polβ and DNA ligases (LIGI and LIGIIIα) execute the repair pathway coordination. Our studies will also
elucidate whether a defective scaffolding function of XRCC1 and cancer-associated polβ variants with altered
BER functions could be determinants of defective pathway coordination. In Project 2, using X-ray
crystallography and cryo-EM, we will elucidate the features of DNA substrate and ligase interaction that dictate
accurate versus mutagenic outcomes during final nick sealing step at atomic resolution and investigate the large
BER multi-protein complexes scaffolded by XRCC1 to gain a novel insight into the structural architecture of the
repair pathway coordination. This equipment supplement requests the purchase of the Nikon Ti2-E
microscope equipped with the fully automated, 4 color H-TIRF module, as well as the epi-fluorescence
module, to visualize BER coordination and to measure DNA ligase activities at the single-molecule level.
This purchase will directly support the experiments in Projects 1 and 2 of our parent proposal. The single
molecule approach using fluorescently labeled repair proteins is highly complementary to the ensemble
experiments that we are currently. We will define the dynamics of BER pathway coordination and provide a more
comprehensive view of the polβ/ligase interplay scaffolded by XRCC1 than either approach alone would give,
thus strengthening the impact of our project. Gaining an understanding of how DNA damage is coordin...

## Key facts

- **NIH application ID:** 10797226
- **Project number:** 3R35GM147111-02S1
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** MELIKE CAGLAYAN
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $200,000
- **Award type:** 3
- **Project period:** 2022-08-15 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10797226

## Citation

> US National Institutes of Health, RePORTER application 10797226, DNA ligase activities during base excision repair coordination (3R35GM147111-02S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10797226. Licensed CC0.

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