A Mutant of PCNA that Exhibits Decreased Binding to Human DNA Polymerase Delta

Abstract

DNA polymerase delta (Pol δ) has at least four subunits: p125, p50, p68, and p12. Pol δ interacts with PCNA, a DNA sliding clamp, in order to achieve the processivity necessary for chromatin replication. Crystallographic studies have shown that p21 binds to the interdomain connector loop (IDCL) of PCNA by the PIP-box as well as to a small and a large hydrophobic pocket. We show here that the D29K mutation in the small hydrophobic pocket of PCNA affects its ability to stimulate Polδ activity when assayed on a polydA/oligodT template primer. Kinetic analyses showed that the maximal activity of Pol δ was not affected, but the KD for PCNA-D29K increased to 82 nM from 27nM for wt PCNA, indicating a weakened binding to Pol δ. By contrast, the binding of D29K PCNA to the three subunit Pol δ (p125/p50/p12) that lacks the p68 subunit was not affected. This suggested that it is the absent p68 subunit that is affected by the mutation. ELISA, and Ni-NTA pull-down assays confirmed that the D29K PCNA exhibits a decreased binding affinity for His-tagged p68. These results suggest that the N-terminal small hydrophobic pocket of PCNA plays a significant role for the functional interaction of PCNA and Pol δ, and may reflect its involvement in the binding of the p68 subunit. (This work was supported by NIH Grant GM31973)