# The molecular basis of SRAP domain DNA-protein crosslinking

> **NIH NIH F31** · VANDERBILT UNIVERSITY · 2021 · $30,396

## Abstract

PROJECT SUMMARY
 Abasic (AP) sites are one of the most common DNA lesions in cells and lead to numerous detrimental
outcomes. Many environmental toxins, including potassium bromate and nitrosamines, lead to DNA base
modification, ultimately inducing the formation of AP sites. AP sites exhibit tendency to form strand breaks, block
transcription, and block DNA polymerases during replication. It is imperative for cells to have mechanisms to
deal with these ubiquitous lesions. AP sites formed outside of S-phase are repaired via the base excision repair
(BER) pathway. AP sites encountered during DNA replication are bypassed by translesion DNA synthesis (TLS)
in an error-prone manner. Recently, a second, error-free pathway for processing replication-associated AP sites
was discovered to involve the protein 5-hydroxymethlycytosine binding, embryonic stem cell-specific (HMCES).
HMCES is important to human health and is dysregulated in aging and cancer. HMCES contains an SOS
Response Associated Peptidase (SRAP) domain, which is conserved across all domains of life. The HMCES
SRAP domain forms a unique, stable DNA-protein crosslink (DPC) to AP sites in single stranded DNA (ssDNA).
This activity forms the basis of a novel DNA repair pathway whereby SRAP protects AP sites from error-prone
polymerases and nucleases during replication. However, the molecular basis for the stability of the DPC is not
yet well-characterized. In collaboration with the Cortez lab at Vanderbilt, a 1.6-Å crystal structure was determined
of the DPC formed between the E. coli SRAP protein, YedK, and ssDNA containing an AP site. This structure
reveals an unprecedented native activity of a protein whereby a highly stable thiazolidine linkage is formed
between the ring-opened form of the AP site and the invariant N-terminal Cys2 residue of SRAP. The structure
also suggests that SRAP has a specificity for DNA structures that would be present at stalled replication forks.
This structure will be utilized to decipher the chemical and physical nature of formation and resolution of this
novel DNA-protein chemical linkage by probing the structure and chemical biology of the SRAP-AP-DPC at the
atomic level. Aim 1 will elucidate the SRAP crosslinking mechanism and Aim 2 will investigate SRAP-DPC
formation in the context of DNA replication structures. Given the importance of AP site repair in response to
environmental toxins as well as cancer and aging, a detailed understanding of SRAP AP site crosslinking is
crucial for understanding how SRAP proteins provide a completely unexplored pathway critical for human health.

## Key facts

- **NIH application ID:** 10312706
- **Project number:** 5F31ES032334-02
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Katherine Amidon Paulin
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $30,396
- **Award type:** 5
- **Project period:** 2020-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10312706

## Citation

> US National Institutes of Health, RePORTER application 10312706, The molecular basis of SRAP domain DNA-protein crosslinking (5F31ES032334-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10312706. Licensed CC0.

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