Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase δ (pol δ). Due to the functional and structural similarity with the β-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol δ. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys254 to Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol δ. Nine basic amino acids that are essential for activating DNA synthesis by pol δ are positioned at the internal α-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the β-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol δ DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the β-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol δ. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology