Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.
AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.
Molecular and Cellular Biology Graduate Program, University of Washington School of Medicine, Seattle, Washington, United States of America.
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Molecular and Cellular Biology Graduate Program, University of Washington School of Medicine, Seattle, Washington, United States of America; Departments of Immunology, Biochemistry and Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America.
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D055162:Biocatalysis; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003564:Cytidine Deaminase / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D010766:Phosphorylation; D059748:Proteolysis; D025801:Ubiquitin / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D025801:Ubiquitin; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase