Studies of proliferating cell nuclear antigen and its role in translesion synthesis

DNA damage on the template strand blocks replication by classical DNA polymerases. One major pathway to overcome these replication blocks is translesion synthesis, which is the replicative bypass of DNA damage by non-classical polymerases. For the cell to utilize translesion synthesis, the non-classical DNA polymerase must be recruited to sites of DNA damage and a polymerase switch must occur between the stalled classical polymerase and the incoming non-classical polymerase. This switching event is believed to be mediated by the replication accessory factor p&barbelow;roliferating c&barbelow;ell n&barbelow;uclear a&barbelow;a&barbelow;ntigen (PCNA). In vivo studies have shown that interactions between PCNA and the non-classical polymerase are required for translesion synthesis. However the regions of PCNA important for the protein-protein interactions between the non-classical polymerase and PCNA are largely unknown. Moreover, in response to DNA damage PCNA is monoubiquitinated at Lys-164. This monoubiquitinated form of PCNA (Ub-PCNA) is required for translesion synthesis. However, the function of monoubiquitinated PCNA in translesion synthesis remains unknown. This is partly because of the difficulty in obtaining sufficient quantities of monoubiquitinated PCNA for biochemical and biophysical studies.;To better understand the role of PCNA during translesion synthesis, I biochemically and structurally characterized two PCNA mutant proteins that are deficient in translesion synthesis: the G178S and E113G PCNA mutant proteins. The structures of both mutant proteins were determined crystallographically and revealed that an extended loop, called loop J, has shifted its position relative to that in the wild type PCNA structure. Steady-state kinetic studies showed that, in contrast to wild type PCNA, which stimulates the non-classical polymerases, the two PCNA mutant proteins failed to stimulate the activity of the non-classical polymerase pol eta. These results indicate that loop J in PCNA plays an essential role in facilitating translesion synthesis.;During the structural studies of the E113G PCNA mutant protein, I observed a unique PCNA structure that failed to form the characteristic PCNA ring shaped structure, through traditional intersubunit interactions of domain A and domain B on neighboring subunits. Instead this non-trimeric PCNA structure formed A-A and B-B intersubunit interactions. The B-B interface is structurally similar to the A-B interface observed for the trimeric ring shaped form. In contrast, the A-A interface is stabilized by hydrophobic interactions. The location of the E113G substitution is directly within this hydrophobic surface and would not be favorable in the wild type protein. This suggests that the side chain of Glu-113 promotes trimer formation by destabilizing these possible alternate subunit interactions.;To better understand the role of Ub-PCNA during translesion synthesis, I developed an Ub-PCNA analog by splitting the protein into two self-assembling polypeptides. This analog supports cell growth and translesion synthesis in vivo. Steady state kinetics studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol eta in vitro. The X-ray crystal structure of this Ub-PCNA analog showed that the ubiquitin moieties are located on the back face of PCNA and interact with it via their canonical hydrophobic surface. Surprisingly, the attachment of ubiquitin does not change the conformation of PCNA. This implies that ubiquitination does not cause an allosteric change in PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases. This is consistent with a "tool belt" model of DNA polymerase exchange, whereby both classical and non-classical polymerases bind to Ub-PCNA simultaneously.