Tabagari, N. | bioRxiv Science

bioRxiv2026

A p53-dependent FBXO44-RAD18 axis limits mutagenesis by terminating translesion DNA synthesis

DNA lesions continually challenge genome replication and threaten genome integrity. DNA damage tolerance pathways, including translesion DNA synthesis (TLS), allow cells to bypass lesions and prevent stalled forks from collapsing into double-strand breaks. Because TLS polymerases are intrinsically error-prone, however, this pathway must be tightly restrained; persistent or deregulated TLS can increase mutagenesis, create therapeutic vulnerabilities, and promote aggressive cancer phenotypes. Through integrated transcriptional profiling, genome-wide CRISPR/Cas9 screening for replication-stress sensitivity, and complementary proteomic analyses, we identify F-box protein 44 (FBXO44) as a late p53-responsive regulator of the TLS mediator RAD18. FBXO44 promotes RAD18 ubiquitination during recovery from replication stress and facilitates shutdown of RAD18-dependent PCNA monoubiquitination. Consistently, FBXO44 loss delays resolution of replication stress and TLS signaling, increases mutation frequency, and is associated with elevated mutational burden and therapy resistance in experimental models and patient datasets. These findings define a p53-FBXO44-RAD18 regulatory axis that limits mutagenic TLS and helps safeguard genome integrity after replication stress.

cancer biology↗

bioRxiv2024

Indel-driven evolution of the canavanine tRNA-editing deacetylase enzyme CtdA

AbstractProteins are heteropolymers composed of twenty standard amino acids. However, over 500 non-proteogenic amino acids exist in nature that can get misincorporated into proteins. Canavanine is an antimetabolite of L-arginine, with which it shares high chemical similarity. It can be utilized by bacteria such as Pseudomonas canavaninivorans in the legume rhizome as a sole source of carbon and nitrogen. However, canavanine is also incorporated in proteins of this bacterium as its arginyl-tRNA synthetase loads tRNAArg with both canavanine and arginine. The recently discovered canavanyl-tRNAArg deacetylase (CtdA) removes canavanine from misloaded tRNAArg and thereby prevents its incorporation in proteins. CtdA is the first enzyme known to edit tRNA mischarged with a non-proteinogenic amino acid. We have elucidated its crystal structure to 1.5 [A] resolution and studied its active site using site-directed mutagenesis. We found that CtdA is a small monomeric enzyme that presents a central, deep cavity that predictably constitutes the canavanine binding site and a positively charged surface area that likely coordinates the CCA-3 tRNA attachment sequence. The stand-alone, trans-editing CtdA is distantly related to the B3/B4 cis-editing domains of the large multi-subunit enzyme Phenylalanine tRNA synthetase (PheRS). Our comparative study reveals that CdtA and B3/B4 domains from bacterial and archeal/eukaryotic origin are three subclasses of a same conserved 3D-fold that differ in type-specific indels, which distinctly shape the substrate binding cleft of these proteins. We propose a unifying nomenclature of secondary structure elements for this 3D-fold. In CtdA, residues E191, Y104, N105 and E118 prove to be relevant for catalysis, of which N105 is conserved in bacterial B3/B4 domains. No other shared residues of catalytic relevance could be identified across enzymes of this class, so that a shared mechanism of catalysis appears unlikely in these editing enzymes.

biochemistry↗