Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA synthesis

The rate of DNA synthesis is crucial for full DNA duplication. We report a key role of p21 in controlling this rate. During normal replication, p21 promotes nascent DNA synthesis alongside the Pol iota ()/p53 complex. When p21 is downregulated but detectable, nascent DNA tracks are longer, discontinuous, and rely on PrimPol. With the complete elimination of p21, nascent DNA tracks become shorter, continuous, and depend on Pol kappa (). Endogenous p21 levels are critical for genomic stability, as both PrimPol and Pol-mediated synthesis can induce chromosomal instability. The residual expression of p21 in p53 null cells influences the involvement of PrimPol or Pol in nascent DNA synthesis and subsequent chromosomal instability. Our results demonstrate that endogenous levels of p21 in cycling cells, insufficient for cyclin-dependent kinase inhibition, prevent genomic instability through PCNA binding, limiting PrimPol and Pol's role in nascent DNA synthesis.