ANCIENT SRAP DOMAIN SHIELDS ABASIC SITES TO PROMOTE GENOME INTEGRITY

Abstract

Abasic (AP) sites are one of the most common DNA lesions that can block replicative polymerases. Currently, all known mechanisms for AP site repair function within the context of double-stranded DNA (dsDNA). A newly discovered pathway dependent on the evolutionarily conserved protein HMCES (5- hydroxymethylcytosine binding embryonic stem cell-specific) provides an alternative to AP site repair in the context of single-stranded DNA (ssDNA). HMCES contains a SOS Response Associated Peptidase (SRAP) domain that is conserved through all domains of life including the E. coli ortholog YedK. HMCES deficient cells are sensitive to DNA damaging agents that generate AP sites and are defective in AP site repair. HMCES shields AP sites by forming a stable DNA-protein crosslink (DPC) intermediate. This intermediate is formed in cells, increases in abundance in response to AP site inducing agents, and is resolved over time by a mechanism that is partially proteasome-dependent. The HMCES-DPC shields AP sites from endonucleases and error-prone polymerases to promote genome integrity. However, the biochemical mechanism of HMCES-DPC formation is not known. To determine the chemical mechanism of SRAP-DPC formation, I characterized the biochemical activities of HMCES and yedK. A 1.6 Å crystal structure shows that SRAP proteins crosslink to AP sites via a stable thiazolidine DNA-protein linkage. This unique linkage is formed by the alpha-amino and sulfhydryl substituents of the N-terminal cysteine of SRAP and the aldehyde form of an AP site. The SRAP-DPC is stable and resistant to cleavage by endonucleases. Furthermore, the covalent linkage is solvent inaccessible. The SRAP-AP DNA structure reveals that HMCES has specificity for AP sites in ssDNA and at junction structures found when replicative polymerases stall at an AP site lesion. A single SRAP domain protein exists in organisms in all three domains of life indicating a critical function. The stability of the SRAP-AP DNA crosslink and unique thiazolidine DPC supports the conclusion that these proteins act to maintain genome stability during DNA replication.