Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4Cdt2‐mediated proteolysis

PCNA links Cdt1 and p21 for proteolysis by Cul4‐DDB1‐Cdt2 (CRL4Cdt2) in the S phase and after DNA damage in mammalian cells. However, other PCNA‐interacting proteins, such as ligase I, are not targets of CRL4Cdt2. In this study, we created chimera constructs composed of Cdt1 and ligase I and examined how the proteolysis of PCNA‐interacting proteins is regulated. Consistent with a recent report using the Xenopus egg system (Havens & Walter 2009), two amino acid elements are also required for degradation in HeLa cells: TD amino acid residues in the PIP box and the basic amino acid at +4 downstream of the PIP box. In addition, we demonstrate that a basic amino acid at +3 is also required for degradation and that an acidic amino acid residue following the basic amino acids abolishes the degradation. Electrostatic surface images suggest that the basic amino acid at +4 is involved in a contact with PCNA, while +3 position extending to opposite direction is important to create a positively charged surface. When all these required elements were introduced in ligase I peptide, the substituted form became degraded. Our results demonstrate that PCNA‐dependent degron is strictly composed to avoid illegitimate destruction of PCNA‐interacting proteins.