THE EXPRESSION LEVEL OF THE CYCLIN KINASE INHIBITOR, P21, IN CELLS TRANSITING S-PHASE MODULATES THE RATE OF DNA FORK SPEED TO PREVENT GENOMIC INSTABILITY

CALZETTA, NICOLAS; MANSILLA, SABRINA; BERTOLIN, AGOSTINA; CAIMI, LILEN; WEISMUELLER, LISA; GOTTIFREDI, VANESA

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Cell cycle arrest outside S phase requires the expression of high levels of p21, a cyclin kinase inhibitor. In opposition, during S- phase, cyclin kinases activity is high due to low levels of p21. Therefore, basal levels of p21 were considered to have no role in S- phase until recently when two manuscripts published by us and another laboratory showed that such low levels of p21 in S-phase modulate the speed of nascent DNA synthesis. Both reports concluded that such an effect of p21 on DNA replication depends on its interaction with PCNA (Proliferating Cell Nuclear Antigen), an auxiliary factor that recruits DNA polymerases to DNA. Intriguingly, however, the two manuscripts indicated opposite effects of p21 depletion on the speed of nascent DNA synthesis (decrease vs increase). Here we show that the extent of p21 downregulation explains such apparently contradictory results. Full p21 downregulation achieved by p21 knockout or efficient siRNA-mediated knockdown decrease fork speed in a manner that depends on the enhanced participation of the low processivity DNA polymerase, Pol Kappa, in DNA replication. In contrast, partial p21 downregulation by siRNA-mediated knockdown increases fork speed by enabling the participation in DNA replication of PrimPol, a DNA polymerase with primase activity that promotes DNA replication restart. Remarkably, both the excess contribution of Pol Kappa or PrimPol to DNA replication events when p21 is fully or partially downregulated trigger the augmentation of several genomic instability markers. Furthermore, our work points to the sources of genomic instability when p21 is fully or partially down regulated being different, and they depend on Pol Kappa and PrimPol activity, respectively. We also identified that transcriptional modulation of p21 as a consequence of loss of p53 on established p53-deficient cell lines triggers genomic instability dependent on Pol Kappa or PrimPol if the basal p21 level is non-existent or low, respectively. Such results reveal an unexpected and quite complex tumor suppressor role of p21 as a guardian of the quality of newly synthesized DNA that prevents spontaneous mutation events.