# Structural determinants of Pol theta function

> **NIH NIH P01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2020 · $322,256

## Abstract

PROJECT SUMMARY
 This project will investigate mammalian DNA polymerase θ, the defining enzyme for repair of DNA
double-strand breaks by polymerase theta-mediated end joining (TMEJ). Despite the biological importance of
TMEJ and its relevance to cancer, we know surprisingly little about its molecular mechanisms. Pol θ is a large
protein (290 kDa in mammalian cells) with a distinctive arrangement of a helicase-like domain linked to a DNA
polymerase domain. In addition to the polymerase domain (PolD), Pol θ possesses a helicase-like domain
(HelD) and a connecting central domain (CenD).
 Structural information is essential for analyzing DNA polymerase mechanisms, especially for a large
multi-domain enzyme such as Pol θ. We determined the first crystal structure of the polymerase domain (PolD)
of Pol θ, together with DNA and an incoming nucleotide. The structure revealed unique features, which help
explain some of the properties of the polymerase. We located 5 insertion loops in the polymerase and pseudo-
exonuclease domains. We presented evidence for dimerization of the PolD, which could function during joining
of two DNA molecules.
 This project aims to fill several major gaps in knowledge, needed to help us understand the unique
activities and structure of Pol θ: (1) What role do the “insertion” loops play in mammalian Pol θ (2) What is the
structural basis of helicase-like domain (HelD) function? (3) How does end-trimming occur during
microhomology selection? (4) How do molecules of Pol θ coordinate repair, using specific interfaces? In
addition to gaining a fundamental understanding of the TMEJ mechanism, the research will reveal new
targeting opportunities for Pol θ inhibition in cancer therapy.
 These structural studies will be highly coordinated within the Program Project with the other three
Projects with complementary experimental approaches – single-molecule characterization of molecular
function, activity assays using full-length proteins, and cellular studies of repair. Substrates, proteins, and
experiments will be designed with Projects 1, 2, and 4 and monitored with feedback via Core A. Protein
purification will be supported by Core B, and cell line construction by Core C. Our combined work will provide
unparalleled insight into the TMEJ pathway, and how its mechanism impacts its ability to fulfill its biological
role. These insights will be critical to our understanding of the contribution of this pathway to genome instability
and carcinogenesis, as well as the evaluation of this pathway as a safe and effective target for cancer therapy.

## Key facts

- **NIH application ID:** 9934673
- **Project number:** 1P01CA247773-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Sylvie Doublie
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $322,256
- **Award type:** 1
- **Project period:** 2020-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9934673

## Citation

> US National Institutes of Health, RePORTER application 9934673, Structural determinants of Pol theta function (1P01CA247773-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9934673. Licensed CC0.

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