# Assembly and Dynamics of Molecular Machines in Genome Maintenance

> **NIH NIH R35** · UNIVERSITY OF IOWA · 2021 · $172,500

## Abstract

ABSTRACT
To maintain stable genomes, cells carry out an accurate and timely replication program and repair such
deleterious DNA lesions as double-stranded breaks, inter-strand crosslinks, and damaged replication forks.
Project 1 of the parent NIH R35GM131704 MIRA grant (PI: Spies) investigates the molecular machinery of
homologous recombination (HR), a cellular process that provides the most accurate means to repair of these
deleterious DNA lesions and damaged replication forks, and thereby contributes to genome stability in normal
cells, but also helps cancerous cells to develop resistance to radiation and DNA-damaging chemotherapy. We
are building a quantitative description of the central step in HR and its regulation, which will draw on the
importance of protein plasticity and conformational dynamics in molecular recognition. Project 2 investigates
multipurpose DNA repair helicases and their ability to coordinate DNA replication through difficult to replicate
regions, thus also contributing to genome stability. Both projects utilize single-molecule total internal reflection
fluorescence microscopy (smTIRFM) to visualize and quantify the dynamic assembly and remodeling of the
nucleoprotein complexes coordinating HR and processing of alternative DNA structures. Mass photometry is a
powerful new technique that will add a new dimension to our measurements by allowing us to quantify the
distributions of the molecular species constituting nucleoprotein complexes.
This application requests funds for acquisition of the Refeyn OneMP Mass Photometer, an instrument that
uses interferometric scattering (iSCAT, aka iScaMS) for label-free detection of molecular mass of individual
macromolecules in solution. This system will facilitate our capacity to perform the experiments proposed in the
parent R35 award and will significantly enhance the rigor of our approaches. Mass photometry will be highly
complementary to smTIRFM analyses, as it will allow us to directly assess the distributions of oligomeric states
of the RAD51 recombinase and its mutants with altered RAD51-RAD51 interface at low, physiologically
relevant concentrations we use in the smTIRFM studies. We will be able to quantitatively evaluate how the
oligomeric states of RAD51 and heterogeneity of the RAD51- and RPA-containing complexes change with
changing the solution conditions (including conditions permitting and restricting ATP hydrolysis, and the
presence of small molecule inhibitors that may affect the RAD51-RAD51 binding affinity). We will be able to
unambiguously determine the equilibrium dissociation constants for and composition of complexes containing
RAD51 and the RAD51-interacting fragments of the tumor suppressor BRCA2, and will follow the formation of
the RAD51 nucleoprotein filament in a manner complementary to our smTIRFM measurements. Addition of the
single-molecule iSCAT measurements will help us to build a completely new picture of the nexus between
perturbations of the RAD51 mo...

## Key facts

- **NIH application ID:** 10377656
- **Project number:** 3R35GM131704-03S1
- **Recipient organization:** UNIVERSITY OF IOWA
- **Principal Investigator:** Maria Spies
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $172,500
- **Award type:** 3
- **Project period:** 2019-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10377656

## Citation

> US National Institutes of Health, RePORTER application 10377656, Assembly and Dynamics of Molecular Machines in Genome Maintenance (3R35GM131704-03S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10377656. Licensed CC0.

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