# Single-molecule studies of Theta mediated end joining

> **NIH NIH P01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2022 · $381,468

## Abstract

PROJECT SUMMARY
This project will investigate mammalian DNA polymerase θ (Pol θ), the defining enzyme for repair of DNA
double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Project 4 (“Single-molecule
studies of TMEJ”) which is part of a Program Project titled, “Polymerase theta, genome instability, and cancer”.
Despite the biological importance of TMEJ, we know surprisingly little about its molecular mechanism and how
defects in the process confer specific vulnerabilities in tumors. Pol θ is a large protein (290 kDa in mammalian
cells) with a distinctive arrangement of a DNA polymerase domain, a helicase-like domain, and a connecting
central domain.
 This project aims to fill several fundamental gaps in our knowledge of TMEJ, and explore novel hypotheses
by employing an array of innovative biochemical, cellular, and single-molecule techniques and assays to define
the key steps and molecular mechanisms of TMEJ. First, we will focus on the initial elusive steps of TMEJ
including synapsis and DNA microhomology search process, and how it is modulated by other repair factors.
Second, we will define the kinetics and regulation of TMEJ during cellular DSB repair. Third, we will establish
how TMEJ contributes to repair of collapsed replication forks and repair of replication conflicts at secondary
DNA structures.
 In Aim 1 “Mechanism of TMEJ synapsis via biochemically reconstituted system” we will establish the
specific contributions of Pol θ helicase, polymerase and other structural domains for initial strand pairing
activity, micro-homology search, and crosstalk with NHEJ and HR
 In Aim 2, “Interplay of TMEJ with NHEJ at DSB sites in cells”, we will measure the specific modes of
recruitment-exclusion and organization of TMEJ repair intermediates as a function of DDR and canonical DSB
repair, and how they are affected by key repair deficiencies.
 In Aim 3, “TMEJ role(s) in repair of collapsed replication forks”, we will investigate the roles of Pol  in repair
of single-ended DSBs (seDSB) formed at collapsed replication forks and resoltuon of toxic secondary
structures.
 The research work will be highly coordinated within the Program Project with the other three Projects
and the three Cores. Our combined diverse approaches include molecular biology, biochemistry, structural
biology, and biophysics. Substrates, proteins, and experiments will be designed with Projects 1, 2, and 3, and
will be constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell
line construction by Core C.

## Key facts

- **NIH application ID:** 10468632
- **Project number:** 5P01CA247773-03
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Eli Rothenberg
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $381,468
- **Award type:** 5
- **Project period:** 2020-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10468632

## Citation

> US National Institutes of Health, RePORTER application 10468632, Single-molecule studies of Theta mediated end joining (5P01CA247773-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10468632. Licensed CC0.

---

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