# DNA Polymerases in Nonhomologous End Joining

> **NIH NIH U01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2020 · $366,253

## Abstract

Repair of chromosome breaks by Nonhomologous end joining (NHEJ) determines the effectiveness of many
cancer therapies, and is essential for assembly of antigen receptor genes (immunoglobulins and T cell
receptors) required for adaptive immunity. This proposal addresses if polymerases specifically to NHEJ
primarily add RNA in the course of this DNA repair pathway - a violation of the central dogma of molecular
biology. Aim 1 experiments address the extent RNA is incorporated during general double strand break repair,
as well as whether RNA incorporation is regulated by cell cycle stage. Cas9 nuclease will be used to target
breaks to a site in chromosome 6. The fraction of RNA in products of repair of this break will be determined by
sequencing products before and after specific destruction of products with embedded RNA. Cells in different
cell cycle phases will be purified, and the fraction of RNA in repair products in these different populations
compared to determine if cell cycle phase influences RNA incorporation. Aim 2 experiments will investigate
why RNA is incorporated during NHEJ. They will further explore preliminary data arguing RNA incorporation by
NHEJ polymerases is required to resolve a subset of broken ends by the NHEJ ligase. The nucleotide added
by NHEJ polymerases will be systematically varied, and effects on ligation assessed. This will address if some
nucleotide analogs – many of which are already used in the clinic for chemotherapy and as antivirals -stimulate
ligation, while other nucleotide analogs have the opposite effect (poisoning of ligation). We will also determine
if the poisoning of NHEJ's ligation step by these analogs could be used to manipulate the response to radiation
therapy. Aim 3 addresses the consequences of the RNA that is now embedded in the repaired chromosome.
RNA in genomic DNA is rapidly replaced with DNA by ribonucleotide excision repair (RER): Aim 3 experiments
will address what mechanisms help prevent RER from re-breaking chromosomes with embedded RNA. This
proposal thus seeks to determine if we must reconsider how much of NHEJ functions, and addresses novel
biological and potentially clinically significant consequences of this new mechanism.

## Key facts

- **NIH application ID:** 9970210
- **Project number:** 5U01CA097096-17
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** DALE A RAMSDEN
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $366,253
- **Award type:** 5
- **Project period:** 2003-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9970210

## Citation

> US National Institutes of Health, RePORTER application 9970210, DNA Polymerases in Nonhomologous End Joining (5U01CA097096-17). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9970210. Licensed CC0.

---

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