# Molecular mechanisms of pathway choice in DNA double strand break repair

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2024 · $368,346

## Abstract

Project Summary
DNA double strand breaks (DSBs) are a particularly toxic form of DNA damage. Even a single DSB can lead to
cell death. Our cells use a number of intricate repair pathways to repair DSBs. For the majority of breaks that
occur in our cells a pathway known as non-homologous end joining (NHEJ) is used to effectively “glue” the
broken strands back together. Alternatively, a strand at each DNA end is “chewed back” in a process known as
resection which generates single-stranded DNA overhangs. Multiple pathways act on these overhangs
including homologous recombination (HR) and microhomology mediated end joining (MMEJ). HR is an intricate
mechanism which uses a sister chromatid to direct repair in a high-fidelity manner while MMEJ is a mutagenic
pathway whose mechanism more closely resembles NHEJ. Proper selection of these repair pathways is critical
for human health as misuse is correlated with gross chromosomal changes that can result in cancer. Often
cancer cells are deficient in DNA repair pathways which allows them to rapidly acquire traits not normally
associated with healthy cells. These repair deficiencies also make them vulnerable to targeted cancer
therapies.
 This proposal seeks to develop a better molecular understanding of how cells choose between these
different DSB repair pathways. Experiments in cells have identified a number of proteins that play a role in this
molecular decision-making process, but we lack an understanding of how these proteins work together.
Traditionally, such knowledge is gained by purifying individual proteins and combining them together to
reconstitute a biological process. However, given the sheer number of proteins, it is currently untenable to take
such an approach. We have recently validated a cell-free extract made from the eggs of the frog Xenopus
laevis as system that recapitulates DSB pathway choice. Combining this physiological biochemical system with
powerful imaging approaches to study the dynamics of DNA DSB repair proteins at the single-molecule level,
we will elucidate the molecular basis of DSB repair pathway choice. Specifically, we will work to clarify how the
NHEJ factor Ku, which antagonizes DNA end resection, is removed from DNA ends (Aim 1); and how the
MMEJ polymerase Pol θ competes with long-range resection on partially resected overhangs (Aim 2). Finally,
we will elucidate how the multi-functional Pol θ uses its diverse enzymatic activities to synapse DNA ends and
search for microhomology (Aim 3). Our studies will reveal significant new insights into DSB repair pathway
selection and regulation. These insights may enable novel ways to alter the balance between these repair
pathways which could have applications in gene editing or in therapies for cancers deficient in DSB repair.

## Key facts

- **NIH application ID:** 10835992
- **Project number:** 5R01CA272436-03
- **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:** $368,346
- **Award type:** 5
- **Project period:** 2022-06-15 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10835992

## Citation

> US National Institutes of Health, RePORTER application 10835992, Molecular mechanisms of pathway choice in DNA double strand break repair (5R01CA272436-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10835992. Licensed CC0.

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