# Structural Biology of the DNA Replication Stress Response

> **NIH NIH R35** · VANDERBILT UNIVERSITY · 2022 · $553,594

## Abstract

PROJECT SUMMARY
The DNA replication machinery is constantly challenged by impediments that stall the replication fork. Replication
stress and stalled forks are a major source of genomic instability, which underlies a number of diseases including
cancer. Replication-repair pathways known as the replication stress response serve to stabilize and restart
damaged forks. However, the molecular mechanisms of these pathways are poorly understood, in part because
of a dearth of structural information for the proteins involved. Our long term goal is to understand the molecular
mechanisms of the replication stress response and how the pathways are interconnected to ensure faithful
completion of DNA replication. Our strategy is to couple structural information of the enzymes and multi-protein
complexes operating at the replication-repair interface with their biochemistry and cellular functions. We are
currently focused on three poorly understood activities at stalled forks—(1) fork reversal and template switching
as a mechanism to stabilize damaged forks and restart replication, (2) protection of labile abasic (AP) sites from
strand cleavage or mutagenic bypass, and (3) priming of DNA synthesis. Fork reversal by the ATP-dependent
DNA translocases HLTF, SMARCAL1, and ZRANB3 involves remodeling of stalled fork into four-way junctions
to prevent fork collapse and facilitate replication restart. Our work will address critical gaps in knowledge related
to how these enzymes provide unique repair activities at damaged forks, their mechanisms of fork reversal, and
how the ubiquitin ligase and DNA remodeling activities of HLTF are coordinated and regulate fork reversal in
cells. Secondly, we are working to understand how the SOS Response Associated Peptidase (SRAP) protein
HMCES forms a stable DNA-protein crosslink (DPC) with AP sites in ssDNA as a means to protect them from
error-prone polymerases and nucleases during replication. AP sites are the most abundant form of DNA damage
and thus it is critical that we understand how cells deal with these potent replication blocks. Our recent structure
of a SRAP DPC forms the basis for further experiments to understand the chemical biology behind this novel
repair pathway. Third, DNA polymerase α-primase (pol-prim) is a core component of the eukaryotic replisome
that initiates de novo DNA synthesis at every Okazaki fragment by synthesizing RNA-DNA primers of defined
length. Despite the importance of this critical activity at the replication fork, its mechanism of action is unknown.
We are addressing this gap in knowledge by trapping complexes of pol-prim with relevant nucleic acid substrates
and intermediates at various stages of its catalytic cycle and visualizing conformational states by electron
microscopy and biophysical approaches. Fundamental knowledge of the conformational dynamics that occur in
pol-prim during de novo DNA synthesis will be the first step toward understanding the coordination of enzymatic
activities...

## Key facts

- **NIH application ID:** 10412932
- **Project number:** 5R35GM136401-03
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Brandt F Eichman
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $553,594
- **Award type:** 5
- **Project period:** 2020-06-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10412932

## Citation

> US National Institutes of Health, RePORTER application 10412932, Structural Biology of the DNA Replication Stress Response (5R35GM136401-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10412932. Licensed CC0.

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