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Ubiquitin-dependent degradation of p21Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress

Milletti, G.; Cadeddu, G.; Adelantado Rubio, A.; Ferraina, C.; Keuper, K.; Munk, S.; De Stefanis, C.; Quacquarini, D.; Rossi, S.; Mastronuzzi, A.; Locatelli, F.; Grumati, P.; Cianfanelli, V.; Nazio, F.; Bartek, J.; Maya-Mendoza, A.; Cecconi, F.

2025-07-31 molecular biology
10.1101/2025.07.28.666126 bioRxiv
Show abstract

The cell cycle is a fundamental process that orchestrates the events that lead to cell replication and division. AMBRA1 is a scaffold factor that interacts with the E3 ubiquitin ligase CRL4-DDB1 complex to regulate the stability of cyclin D-type proteins, key regulators of the G1/S phase transition. However, how cells complete S-phase and coordinate the speed of DNA synthesis is still to be fully elucidated. Here we demonstrate that AMBRA1 affects the turnover of p21Waf1/Cip1 and p27Kip1, by coupling the CRL4-DDB1 complex directly to these proteins. In the absence of AMBRA1, the increased stability of p21Waf1/Cip1, rather than p27Kip1, resulted in the accumulation of replication stress by leaving under-replicated DNA during the S phase. We also found that AMBRA1-depleted cells with consequent high p21Waf1/Cip1 are sensitive to the inhibition of lagging-strand DNA synthesis. Of clinical relevance, low levels of AMBRA1 in Sonic-Hedgehog medulloblastoma correlate with a worse prognosis, suggesting that AMBRA1 expression levels could be an informative criterion for patient stratification and treatment.

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