Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dr. Richard D. Wood

Committee Member

Dr. Kevin M. McBride

Committee Member

Dr. Karen M. Vasquez

Committee Member

Dr. Rick Finch

Committee Member

Dr. Randy Legerski

Abstract

In mammalian cells, DNA polymerase θ (POLQ) is an unusual specialized DNA polymerase whose in vivo function is under active investigation. The protein is comprised of an N-terminal helicase-like domain, a C-terminal DNA polymerase domain, and a large central domain that spans between the two. This arrangement is also found in the Drosophila Mus308 protein, which helps confer resistance to DNA interstrand crosslinking agents. Homologs of POLQ and Mus308 are found in eukaryotes, including plants, but a comparison of phenotypes suggests that not all of these genes are functional orthologs. Flies with defective Mus308 are sensitive to DNA interstrand crosslinking agents, while mammalian cells with defective POLQ are primarily sensitive to DNA double-strand breaking agents. Cells from Polq-null mice are hypersensitive to radiation and the peripheral blood cells of these mice display increased spontaneous and ionizing radiation-induced levels of micronuclei (a hallmark of gross chromosomal aberrations), though the mice apparently develop normally.

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand-breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, the repair of DNA ends with long 3′ single-stranded overhangs was monitored. End joining events that retained much of the overhang were dependent on POLQ, and independent of Ku70. To analyze this repair function in more detail, immunoglobulin class switch joining between DNA segments in antibody genes was examined. POLQ participates in the end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers.

DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. A marked increase in Myc/IgH translocations was observed in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway (termed synthesis-dependent end joining) that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

Class switch and junction analysis was also performed in mice lacking POLN, another DNA polymerase related to POLQ. I observed that POLN does not operate in the same alternative end joining pathway as does POLQ. Loss of Poln does not enhance the DNA damage hypersensitivity seen in cells lacking Polq. These findings suggest that while these two polymerases are structurally related they appear to have distinct functions in the cell. Analysis of the Poln phenotype is still ongoing. Further analysis of POLN and POLQ is required to clarify the mechanism by which they function in the cell.

Keywords

POLQ, DNA polymerase, genome stability, alternative end joining, chromosomal translocation, class switch recombination