The molecular basis of SRAP domain DNA-protein crosslinking

点击翻译按钮获取中文摘要

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.