Characterization of SRAP Domain DNA-Protein Crosslink Formation and Reversal with DNA Abasic Sites
Paulin, Katherine Amidon
Apuirinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form crosslinks to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The protein HMCES protects cells from DNA strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA crosslinks derived from AP sites. The highly conserved SRAP domain of HMCES and its Escherichia coli ortholog YedK mediate lesion recognition by forming a DNA-protein crosslink (DPC) to AP sites in single-stranded DNA (ssDNA). Here we discover the basis of AP site protection by SRAP domains from a crystal structure of the YedK DPC. YedK forms a stable thiazolidine linkage between a ring-opened AP site and the α-amino and sulfhydryl substituents of its N-terminal cysteine residue. I then review the collection of recently reported SRAP crystal structures, which provide a unified basis for SRAP specificity. Despite the importance of HMCES to genome maintenance, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, I show that the enzyme catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. Additionally, I demonstrate that YedK reacts with polyunsaturated aldehydes at DNA 3′-ends generated by bifunctional DNA glycosylases and that the enzyme catalyzes direct reversal of the DPC to regenerate the AP site, providing insight into possible mechanisms by which HMCES DPCs are resolved in cells.