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

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2021 · $349,170

## Abstract

Project Summary
In this proposal we will apply biochemical and single-molecule approaches to understand the mechanism of
non-homologous end joining (NHEJ), the primary DNA double strand break (DSB) repair pathway in human
cells. During NHEJ, core factors, end processing factors and other accessory factors tether DNA ends together
and ultimately ligate them. While the biochemical activities of these individual factors are known to varying
extents, it remains poorly understood how these factors assemble into a synaptic complex and how their
various enzymatic activities are coordinated. To that end, we will apply single-molecule imaging
approaches in Xenopus egg extract to directly follow NHEJ complex formation and end synapsis in
real time during a physiological repair reaction. Completion of the specific aims below will provide an
increased mechanistic understanding of NHEJ, which will aid efforts to therapeutically target NHEJ and to
modulate repair outcomes during CRISPR-Cas gene editing.
Aim 1: How does the synaptic complex assemble and evolve during NHEJ?
Upon DSB formation it is critical that DNA ends are rapidly synapsed so as to prevent the ends from diffusing
apart and joining with the wrong partner. We have shown that paired ends pass through two distinct synaptic
states during repair. Initially ends are held in a relatively unstable long-range synaptic complex before
transitioning to a stable short-range synaptic complex in which the ends are poised to be ligated. In this aim we
will determine the unique sets of intermolecular interactions that characterize the synaptic complexes and
describe how these interactions evolve during repair. In particular, we will elucidate how the core NHEJ factors
XLF, XRCC4 and LIG4 contribute to end synapsis and determine how accessory factors facilitate assembly of
the synaptic complexes.
Aim 2: How do end processing factors gain access to DNA ends?
To minimize aberrant end processing and resection, DNA ends are rapidly bound by Ku and other factors. In
the prior funding period, we showed that even NHEJ-associated end processing is restricted until formation of
the ligation-competent short-range synaptic complex. This regulation prioritizes ligation over error-prone end
processing. In this aim we will elucidate the molecular steps that enable end deprotection and allow for end
processing. Furthermore, we will determine how Ku is remodeled on DNA ends during repair and examine the
consequences on NHEJ by blocking this remodeling. Next, we will determine how different processing factors
compete for DNA ends after they become accessible. Finally, we will apply our mechanistic insight into the
regulation of end processing to decrease the fidelity of repair of CRISPR-Cas9 induced breaks in cells.

## Key facts

- **NIH application ID:** 10164800
- **Project number:** 5R01GM115487-06
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Joseph J. Loparo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $349,170
- **Award type:** 5
- **Project period:** 2015-09-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10164800

## Citation

> US National Institutes of Health, RePORTER application 10164800, Visualizing DNA break repair: single-molecule studies of non-homologous end joining (5R01GM115487-06). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10164800. Licensed CC0.

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