# Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease

> **NIH NIH R01** · WRIGHT STATE UNIVERSITY · 2020 · $300,000

## Abstract

Chromosome breaks are the most dangerous form of DNA damage because they result in multiple types of
mutations and gross chromosome rearrangements. DNA is most sensitive to breakage during replication, when
hard-to-replicate noncanonical DNA structures cause replication fork stalling. Noncanonical DNA structures are
strongly implicated as endogenous sources of chromosome breaks and translocations leading to
developmental defects and cancers, however, the mechanisms by which replication fork stalling causes DNA
double strand breaks (DSBs) are not known.
 Despite significant analyses of DNA damage response proteins in global or single molecule studies where
the sites of damage are not identified, the molecular mechanisms of replication-dependent DNA strand
breakage and repair at specific sites in human cells are incompletely understood. To address this knowledge
gap, we will study two types of natural replication barriers (CTG/CAG trinucleotide repeats and asymmetric
purine-pyrimidine (Pu/Py) mirror repeats) integrated at an ectopic site in the human genome where their
structure and effect on replication can be manipulated. We also examine several endogenous replication fork
barriers that induce DSBs during DNA replication. We will use PCR, DNA sequencing, chromatin
immunoprecipitation, mass spectrometry and flow cytometry to show (1) how polymerase stalling at
noncanonical DNA structures causes DSBs, (2) how DNA repair proteins act to remodel stalled replication
forks to restart synthesis, and (3) the mechanisms and genomic consequences of DSB recombination at
structure-induced fork barriers.
 We will test the hypothesis that noncanonical DNA structures induce DSB by blocking the progress of DNA
polymerases, promoting nuclease-sensitive fork regression, and inhibiting DNA end processing required for
recombination. Conceptual advances from this work will include determination of the molecular mechanisms of
DSB formation near specific stalled forks, biochemical analysis of replication fork reversal, and identification of
how the processing of structure-induced DSB differs that of nuclease-induced `clean' DSB. Our long-term goal
is to define the role of DNA structure-induced g e n o m e instability in human disease.
 Aim 1 will disclose the relationship between fork stalling and damage signaling, the biochemistry of fork
reversal, the function of structure-specific endonucleases at stalled forks, and the impact of DNA secondary
structure on fork resection and repair. Aim 2 will build on our demonstration that the Fanconi anemia type J
protein (FANCJ) is essential for the maintenance of noncanonical DNA structures across the genome during
replication stress, to determine the mechanisms of FANCJ dependent microsatellite stabilization. In Aim 3 we
will characterize the genomic consequences of FANCJ deficiency. Our experiments will show how hard-to-
replicate DNA sequences cause chromosome breaks and mutations that lead to genetic disease.

## Key facts

- **NIH application ID:** 10004155
- **Project number:** 5R01GM122976-04
- **Recipient organization:** WRIGHT STATE UNIVERSITY
- **Principal Investigator:** Michael LEFFAK
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $300,000
- **Award type:** 5
- **Project period:** 2017-09-08 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10004155

## Citation

> US National Institutes of Health, RePORTER application 10004155, Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease (5R01GM122976-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10004155. Licensed CC0.

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