# Assembly and Dynamics of Molecular Machines in Genome Maintenance

> **NIH NIH R35** · UNIVERSITY OF IOWA · 2024 · $206,080

## Abstract

ABSTRACT
Efficient genome maintenance is a double-edged sword. Accurate repair of DNA lesions and damaged
replication forks promotes genome stability, which is a key to avoiding cancer, aging and neurodegenerative
diseases associated with DNA repeat expansion. My research program under the parent NIH R35GM131704
MIRA grant (PI: Spies) investigates the molecular and structural mechanisms by which the intermediates of DNA
metabolism bound by Replication Protein A (RPA) are channeled into DNA repair, protection of DNA replication
forks, homologous recombination, DNA damage tolerance and signaling. Our goal is to reconstitute (in vitro and
in singulo) and manipulate (in vivo) the elaborate network of DNA repair mechanisms, and to understand how
cells “choose” between accurate DNA repair and “rogue” mechanisms that destabilize the genome.
Ability to visualize and investigate biologically important molecules individually, in real time, and under
physiological conditions is critical for developing mechanistic understanding of how cells work and communicate,
how molecular machines assemble and function, and, in general, how physiological functions emerge from the
chaos of stochastic molecular events and interactions. All projects under the parent MIRA award utilize single-
molecule total internal reflection fluorescence microscopy (smTIRFM), correlated optical tweezers and
fluorescence microscopy (CTFM), mass photometry, structural biology (CryoEM) and biochemical
reconstitutions to visualize and quantify the dynamic assembly and remodeling of the nucleoprotein complexes
coordinating DNA repair events, homologous recombination and processing of non-canonical DNA structures.
While my lab has been successful in broadening the scope of state-of-the-art single-molecule approaches to
study macro-molecular interactions, their dynamics and inhibition using smTIRFM, surface-tethering required for
this approach makes it impossible to rapidly screen experimental conditions, build fluorescence and Forster
resonance energy transfer (smFRET) distributions.
This application requests funds for acquisition of the EI-FLEX, a single-molecule fluorescence spectrometer
which has a capacity to simultaneously and rapidly record (i) fluorescence and FRET from single molecules in
solution, and (ii) fluorescence decay correlations which inform on complex sizes. Such information on the
complexes containing RPA, alt-RPA, FANCJ, REV1 and PARP1 will help us to build a completely new picture of
the nexus between RPA configuraiotnal dynamics and shuttling of the RPA-containing complexes into specific
genome maintenance pathways.

## Key facts

- **NIH application ID:** 11095575
- **Project number:** 3R35GM131704-06S1
- **Recipient organization:** UNIVERSITY OF IOWA
- **Principal Investigator:** Maria Spies
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $206,080
- **Award type:** 3
- **Project period:** 2019-04-01 → 2029-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11095575

## Citation

> US National Institutes of Health, RePORTER application 11095575, Assembly and Dynamics of Molecular Machines in Genome Maintenance (3R35GM131704-06S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/11095575. Licensed CC0.

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