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theses

Long Terminal Repeat retrotransposable elements (LTR-TEs) are a large group of eukaryotic Transposable Elements characterized by flanking repeats in tandem orientation - the LTRs. The LTRs of these elements contain sequences that recruit proteins involved in their expression, replication, silencing, organization, and stability. A successful transposable element must maximize its reproductive amplification without jeopardizing its host, and several characterized LTR-TEs appear to accomplish this through the selection of integration sites away from protein coding sequences. However, despite their high relatedness, a universal mechanism that explains how these parasitic elements avoid coding sequences has not been established. Through sequencing of de novo integration sites of the LTR-TE Tf1 from the fission yeast Schizosaccharomyces pombe, we found a strong integration preference for locations near the binding site of Sap1. Sap1 has been previously shown to be a DNA-binding protein that controls the directionality of DNA replication by causing polar fork arrest. Sap1 mutations that mildly affect binding but strongly affect fork barrier activity decrease Tf1 retrotransposon efficiency ten-fold, indicating that Sap1 replication fork barrier activity is a stronger predictor of Tf1 integration than DNA binding. Further, synthetic Sap1 binding sites placed near DNA origins are only competent at Tf1 recruitment when placed in blocking orientation. Interestingly, the fork arresting activity of an independent factor provided in cis can increase the integration efficiency of a barrier-incompetent Sap1 binding site. Thus, both Sap1 binding and replication fork arrest are necessary for Tf1 integration. Together, these data suggest that Sap1 guides insertion of Tf1 by tethering the intasome and blocking the progression of the replication fork, and that the Tf1 transposon uses features of arrested forks to insert into the host genome. Since fork arrest is detectable in many genomic features that recruit LTR-RT integration, such as type III promoters and heterochromatic sequences, these observations point to a universal mechanism for determination of LTR-TE tropism. The questions surrounding the molecular mechanism of Tf1 transposition led to the examination of the CRISPR/Cas9 system as a tool for tethering Tf1 to stalled forks in vivo. However, the CRISPR/Cas9 toolkit had not been developed for S. pombe. Using a novel processed RNA Pol II promoter and the Hammerhead ribozyme we developed a highly efficient CRISPR/Cas9 expression system, leading to >95% modification efficiencies without selection.

ETD_7660

Ph.D.

Includes bibliographical references

by Jake Zachary Jacobs

doi:10.7282/T3668GHK

ETD doctoral

The author owns the copyright to this work.