Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

King, Megan

Abstract

DNA double-stranded breaks (DSBs) represent a danger to genome stability. Cells repair DSBs mainly through one of two pathways: canonical non-homologous end-joining (cNHEJ), which requires the direct ligation of the two DSB ends, or homologous recombination (HR), which involves the use of a homologous template to create a faithful repair product. While HR is often considered to be an error-free mechanism, the ultimate fidelity of this pathway depends on the ability of the cell to identify and engage with the correct homologous template. This is particularly challenging during repair of repetitive regions of the genome for which non-allelic sequences can errantly be used as templates. Cells have evolved multiple mechanisms to ensure genomic stability of repetitive regions; one of which is nuclear compartmentalization. Repetitive regions tend to be clustered in their own compartments within the 3D space of the nucleus. In the fission yeast Schizosaccharomyces pombe, repetitive sequences tend to be heterochromatized and located at the nuclear periphery. Multiple studies across model systems have shown that DSBs at heterochromatic regions will move outside of their heterochromatic compartment to complete repair, suggesting that compartmentalization provides an additional layer of stringency during DNA repair. However, due to the correlation between heterochromatin and the nuclear periphery, these studies did not disentangle whether this effect was due to the silencing effect of heterochromatin or to nuclear compartmentalization. As such, I developed a model to study spontaneous DNA damage and repair that occurs at repetitive protein coding genes of the S. pombe flocculin-like (PFL) family. I found that the genes encoding most members of this protein family reside at the nuclear periphery by virtue of their close proximity to binding sites for the CENP-B like protein, Cbp1. Tethering to the nuclear periphery via Cbp1 enforces the stability of the flocculin genes to both intragenic recombination within the protein repeat-encoding sequence and restrains intergenic recombination between homeologous repeat-encoding sequences. Another mechanism that leverages nuclear compartmentalization to enforce proper DNA repair is the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex is known to enforce proper pairings of homologous chromosomes during meiosis, likely by a kinetic proofreading-type mechanism that will reject non-homologous chromosome pairs. I found that it also enforces the use of proper homologous templates during DNA repair, as loss of the LINC complex component Kms1 enhances the use of microhomology-mediated end-joining (MMEJ) as well as heightened use of a homeologous template, resulting in HR between non-allelic sites. My observations suggest that S. pombe leverages nuclear compartmentalization to maintain genome stability, particularly in repetitive regions. Moreover, association of DSBs with Kms1-containing LINC complexes enforces stringency to both attenuate the use of microhomology that drives mutagenic end-joining or selecting the correct template for HR.

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