FIGURE 4.

Identification of a PCNA residue that is essential for CRL4^Cdt2-mediated destruction. A, an image of the PCNA-p21 co-crystal structure (24) was generated using PDB accession number 1AXC and PyMOL (available on-line). PIP box residues of p21 are shown in green, the B+4 of p21 is shown in blue, Leu-126 and Ile-128 (of the PCNA-79 mutant) are shown in red, and PCNA acidic residues Asp-122 and Glu-124 are shown in purple. B, sequence alignment of the interdomain connector loop of PCNA (amino acids 119–133) from different species. Leu-126 and Ile-128 are shown in red; Asp-122 and Glu-124 are shown in purple. C, PCNA-depleted HSS was supplemented with recombinant human PCNA^WT, PCNA^DE/AA (PCNA^D122A/E124A), PCNA^D122A, PCNA^D120A, or PCNA^E124A. Recombinant Set8^WT, Cdt1^1–243, and 5 ng/μl MMS plasmid were also added to the extract. Reactions were stopped at the indicated times and blotted for Cdt1 (both endogenous and Cdt1^1–243) or Set8. Samples were run on two separate gels that were processed under the same conditions. Additionally, PCNA^WT samples were run on both gels, exposures were matched, and similar exposures were used.