# Visualizing DNA break repair: single-molecule studies of non-homologous end joining

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2024 · $353,955

## Abstract

Project Summary
DNA double strand breaks (DSBs), an extremely toxic form of DNA damage, arise both spontaneously and in a
programmed manner during antigen receptor development. Failure to properly repair these breaks leads to a
variety of deleterious outcomes including cell death and gross chromosomal rearrangements that are a major
driver of cancer. The primary DSB repair pathway in humans is non-homologous end joining (NHEJ). Deficiencies
in NHEJ result in several severe genetic diseases that are characterized by immune deficiency, sensitivity to
ionizing radiation and developmental abnormalities. Conversely overactive NHEJ in tumors provides resistance
to DSB-inducing treatments and is correlated with poor clinical prognosis. Given these observations, inhibitors
of NHEJ are a focus of therapeutical development. To fully exploit the clinical potential of these inhibitors it is
critical to understand the molecular mechanism of NHEJ and how its various steps are regulated.
 During NHEJ, DNA ends are held together by a multiprotein synaptic complex and ultimately ligated back
together. As DNA ends are often not initially compatible for ligation, several NHEJ-associated end processing
enzymes act on ends to enable ligation. Some of these end processing enzymes are error-prone; their activity
must be regulated to minimize genome alterations at the repair junction.
 In this project, we will further elucidate the molecular mechanism of DNA end synapsis and how it is
coordinated with end processing to maximize the fidelity of NHEJ. To do this we will employ novel single-molecule
imaging approaches developed by my laboratory to probe the structural dynamics of the NHEJ machinery in a
physiologically complex cell-free system. These approaches will allow us to observe how DNA ends are brought
together during repair and how protein factors are recruited to the NHEJ machinery and gain access to DNA
ends. We will pursue the following three aims. In Aim 1, we will determine how ligation is coordinated with end
processing within the Short-Range (SR) synaptic complex. Formation of the SR complex requires end binding
of the ligase which likely competes with end processing factors for DNA ends. In Aim 2, we will elucidate how
PAXX contributes to DNA end synapsis. Finally, in Aim 3, we will determine how end processing factors are
regulated within the SR complex. Completion of these aims will provide rich molecular insight into how the NHEJ
machinery carries out DNA end synapsis and processing to properly repair DSBs.

## Key facts

- **NIH application ID:** 10879630
- **Project number:** 2R01GM115487-09
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Joseph J. Loparo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $353,955
- **Award type:** 2
- **Project period:** 2015-09-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10879630

## Citation

> US National Institutes of Health, RePORTER application 10879630, Visualizing DNA break repair: single-molecule studies of non-homologous end joining (2R01GM115487-09). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10879630. Licensed CC0.

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