# Single-molecule organization and kinetics of the NHEJ repair machinery inside the cell

> **NIH NIH F31** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2021 · $46,036

## Abstract

PROJECT SUMMARY
DNA double-stranded breaks (DSBs) are the most toxic DNA lesions, and in mammalian cells
they are predominantly repaired via the non-homologous end-joining (NHEJ) pathway. Upon
formation of a DSB, p-53 binding protein-1 (53BP1), a chromatin modulator, forms higher order
structures (foci) and which act as platforms to recruit DSB repair proteins to damaged chromatin
and promote NHEJ by blocking end resection. Several human syndromes including severe
combined immunodeficiency, and an increased sensitivity to ionizing radiation (IR) and
chemotherapeutic drugs result from mutations in NHEJ proteins. Additionally, deficiencies in
NHEJ may also result in mutagenic alternative end-joining (a-EJ) DSB repair, which is established
in some forms of cancer. Despite much progress in the field, the organization and kinetics of
NHEJ and a-EJ factors remain undefined inside the cell. Furthermore, fundamental mechanisms
of repair foci and their effects on NHEJ remain poorly understood. These gaps are due to inherent
limitations of conventional ensemble methods. Here, I propose to resolve these knowledge gaps
by defining the molecular mechanism of the human NHEJ repair process at the level of individual
53BP1 foci via single molecule techniques such as single-molecule tracking (SMT) and stochastic
optical reconstruction microscopy (STORM). These approaches will provide novel information
that may be applicable to future therapy development of PARP inhibitors and NHEJ-related
diseases such as XLF deficiency syndrome. The aims of the proposal are to (1) establish the
kinetics and organization of NHEJ factors within individual 53BP1 foci, and (2) determine the role
of 53BP1 foci in regulating the kinetics and organization of the DSB repair machinery.

## Key facts

- **NIH application ID:** 10218072
- **Project number:** 5F31GM136110-02
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** Maria Benitez-Jones
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 5
- **Project period:** 2020-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10218072

## Citation

> US National Institutes of Health, RePORTER application 10218072, Single-molecule organization and kinetics of the NHEJ repair machinery inside the cell (5F31GM136110-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10218072. Licensed CC0.

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