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Abstract
The constant battle between viruses and host organisms provides evolutionary pressure for both to evolve powerful defense and counter-defense mechanisms to one another. Many prokaryotes defend themselves from viruses and other mobile genetic elements (MGEs) such as plasmids, using adaptive immune systems called CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated) systems. These CRISPR-Cas systems function by integrating short fragments of DNA sequences (called spacers) from the MGEs into host cell CRISPR genomic arrays to provide a heritable record of the captured MGE sequences (a process called adaptation). Subsequent CRISPR array expression and RNA processing leads to the production of small CRISPR RNAs (crRNAs) that guide recognition and Cas nuclease-mediated destruction of invasive MGE nucleic acids to prevent further MGE infection. While integration of invasive MGE DNA fragments into a host CRISPR array is normally an exceptionally rare event, we observed that introduction of the first natural MGE (the pT33.3 conjugative plasmid) tested for our model organism, the hyperthermophilic archaeon Pyrococcus furiosus, stimulated a strikingly robust spacer acquisition response whereby the majority of cells captured DNA fragments specifically against this pT33.3 plasmid into the host CRISPR arrays. Our investigation revealed that the observed “hyper-adaptation” response was mediated by a specialized pathway known as “primed” adaptation” that relied upon the presence of a partially matching, naturally acquired spacer in one of the CRISPR arrays. In other work described in this thesis, we obtained key mechanistic insight into the important question for how new spacers become integrated in a directional manner at the leader end repeat rather than internal repeats within CRISPR arrays. Here, through a combination of micrococcal nuclease DNA protection assays, in vivo and in vitro adaptation studies and high throughput sequencing, we discovered that the archaeal histones of Pyrococcus furiosus play a major role in the guidance of new spacers to the first repeat of CRISPR arrays. The work in this dissertation highlights the unique response against the first natural MGE for Pyrococcus furiosus and reveals a novel role of archaeal histone proteins in shaping integration of new spacer DNA in a polarized manner at CRISPR arrays.