FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

Ubiquitin linkage is critical in directing the cellular fate of a ubiquitinated protein. Although K48-linked polyubiquitination of p53 leads to its degradation, whether K48-independent ubiquitin linkages are involved in p53 activation remains unknown. Here, we show that FATS acts as a p53 activator by inhibiting Mdm2 binding to p53 and stimulating non-proteolytic polyubiquitination of p53. Knockdown of FATS impairs p53 stabilization and activation in response to DNA damage. Furthermore, the NH2-terminal domain of FATS is sufficient to exhibit ubiquitin ligase (E3) activity and assemble ubiquitin polymers through K11-, K29- and K63-linkages, independently of the ubiquitin-conjugating enzyme (E2). FATS promotes p53-dependent transcription of p21, leading to robust checkpoint response. The E3 activity of FATS is required for promoting p53 stability and activation in response to DNA damage. Our findings reveal K48-linkage-independent non-linear polyubiquitination of p53 as a new barcode for p53 activation.