Abstract
<jats:p>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.</jats:p>