Structural and Functional Studies of ASCC1 in DNA and RNA Alkylation Damage Response

Advisor: John Tainer, PhD

Alkylating agents from environmental, endogenous, and certain chemotherapeutic sources constantly challenge cellular nucleic acids. Repairing the damage caused by alkylation is necessary for maintaining genomic stability. ALKB family Human Homolog ALKBH3 plays a key in repairing alkylated damages in DNA and RNA molecules. Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with splicing, transcriptional regulation, and translational control) and two-histidine phosphodiesterase (PDE) (associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link ASCC1 loss of function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). The Cancer Genome Atlas (TCGA) analysis suggests that ASCC1 RNA overexpression in tumors correlates with poor survival, Signature 3 mutations, and genetic instability markers. We determined the crystal structure of Alvinella pompejana (Ap) ASCC1 with 1.14 A resolution. A comparison of the ApASCC1 structure with the human (Hs) PDE domain reveals conserved features that have been preserved for over 500 million years of evolution. Extending our understanding of the KH domain signature Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two pairs of His-X-Ser/Thr-X (HXT) motifs (X being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. A flexible inhibitory loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Our collective structural, architectural, and bioinformatic results contribute significantly to our understanding of the role of ASCC1 in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Advisory Committee:
John Tainer, PhD, Chair
Alemayehu Alex Gorfe, PhD
John Putkey, PhD
Katharina Schlacher, PhD
Irina Serysheva, PhD