Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Ling, Hong

Abstract

DNA polymerase iota (polι) is a member of the Y-family, polymerases which are key components in translesion synthesis (TLS). As part of the DNA damage response, TLS allows cells to bypass damaged template DNA. Each member of the Y-family is capable of accurately replicating across from certain lesions. All Y-family polymerases are recruited by ubiquitination of the DNA sliding clamp, PCNA, by direct interaction with PCNA and ubiquitin. The mechanism of polymerase choice is not well understood, nor are the interactions between Ub-PCNA and the TLS polymerases. We studied the structure of the complex between the interacting region of polι and Ub-PCNA. Polι appears to be unable to bind all three monomers of homotrimeric Ub-PCNA simultaneously, even in a heavily truncated form. The maximum complex ratio observed was two polymerases per Ub-PCNA ring. This assembly ratio limit may give insight into switching of multiple polymerases at the PCNA platform in DNA damage response.

Summary for Lay Audience

Accurate replication of DNA is a key biological process. Organisms have evolved complementary maintenance processes to maintain the integrity of genetic information, such as low-error replication machinery, damage repair mechanisms, and replacement mechanisms using templates. These all work together to keep the mutation rate low. Translesion synthesis (TLS) is one component of the damage-response process, but its role is different. TLS replication tolerates damaged DNA and allows replication to continue past lesions when repair processes cannot respond in time, at the risk of mutation. TLS must be kept inactive to avoid unnecessary mutations, but quickly activated at damaged sites. This is accomplished by modifying the major hub protein for replication processes, a ring-shaped trimeric protein called PCNA. A small marker protein called ubiquitin (Ub) is added to a specific site, which displaces the regular replication machinery and acts as a binding site for the TLS proteins. The structure of the complex between Ub-PCNA and TLS proteins is poorly understood. Each of the TLS polymerases handles different types of damage well, but all of them are called by adding ubiquitin to PCNA. DNA polymerase iota (polι), one of the TLS proteins, was chosen an example to investigate. After initial attempts at structure determination by crystallography failed, indirect methods of probing the complex were used. The stoichiometry of the complex was found to be limited to two small polι constructs per UbPCNA ring by multiple independent methods. Coarse structural determination was also attempted by small-angle X-ray scattering, with ambiguous results. Here we argue that the limited stoichiometry observed and the unusual organization of polι suggests that its role is that of a final attempt at lesion bypass when other TLS polymerases have failed. This is in line with the minimal effect of having knocked out polι in model organisms, and a reasonably safe role considering its high tendency to mutate and relatively small number of lesions it appears to handle compared to the major TLS polymerase.

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