Date of Degree
PhD (Doctor of Philosophy)
One major pathway to overcome DNA damage induced replication blocks is translesion DNA 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 is recruited to sites of DNA damage, and a polymerase switch occurs between the stalled classical polymerase and the incoming non-classical polymerase. This process requires the replication accessory factor proliferating cell nuclear antigen (PCNA) and its monoubiquitination at Lys-164.
To better understand the role of PCNA during translesion synthesis, I biochemically and structural characterized two PCNA mutant proteins, G178S and E113G PCNA, which are defective in translesion synthesis. The X-ray crystal structure of both mutant proteins showed a shift in an extended loop, called loop J, compared to the wild type PCNA structure. Steady state kinetic studies determined that in contrast to wild type PCNA which stimulates the non-classical polymerases, the two PCNA mutant proteins fail to stimulate the activity of the non-classical polymerase pol η. 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 shape 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 biochemically and structurally characterize the impact of monoubiquitinating PCNA (Ub-PCNA), 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, and steady state kinetic studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol η in vitro. The X-ray crystal structure of Ub-PCNA showed that the ubiquitin moieties are located on the back face of PCNA. Surprisingly, the attachment of ubiquitin does not change PCNA's conformation. This implies that PCNA ubiquitination does not cause an allosteric change to PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases.
DNA replication, PCNA, polymerase, translesion synthesis
ix, 183 pages
Includes bibliographical references (pages 163-183).
Copyright 2010 Bret D Freudenthal