STRUCTURE-FUNCTION RELATIONSHIP OF THE EUKARYOTIC DNA-REPLICATION FACTOR, PROLIFERATING CELL NUCLEAR ANTIGEN

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymera se delta (pol delta), Due to the functional and structural similarity with the beta-subunit of Escherichia coli DNA polymerase III, it has b een proposed that PCNA would act as a molecular clamp during DNA synth esis, By site-directed mutagenesis and biochemical analyses, we have s tudied the functional domains of human PCNA required for stimulation o f replication factor C (RF-C) ATPase and DNA synthesis by pol delta, S hort deletions from either the N or C termini caused drastic changes i n extraction and chromatographic behaviors, suggesting that both of th ese terminal regions are crucial to fold the tertiary structure of PCN A, The short C-terminal stretch from Lys(254) to Glu(256) is necessary for stimulation of RF-C ATPase activity, but not for stimulation of D NA synthesis by pol delta, Nine basic amino acids that are essential f or activating DNA synthesis by pol delta are positioned at the interna l alpha-helices of PCNA, This result is in good agreement with the obs ervation that PCNA has a ring structure similar to the beta-subunit an d clamps a template DNA through this positively charged internal surfa ce, Several other charged amino acids are also required to stimulate e ither RF-C ATPase or pol delta DNA synthesis, Some of them are positio ned at loops which are exposed on one of the side surface of PCNA adja cent to the C-terminal loop. In addition, the beta-sheets composing th e intermolecular interface of the trimeric PCNA are important for inte raction with pol delta, Therefore, the outer surface of PCNA has multi ple functional surfaces which are responsible for the interaction with multiple factors, Furthermore, the two side surfaces seem to be funct ionally distinguishable, and this may determine the orientation of tra cking PCNA along the DNA.