TEMPLATE INDEPENDENT DOUBLE STRAND BREAK REPAIR AND RECOMBINASE MODULATION IN THE ARCHAEON SULFOLOBUS SOLFATARICUS
Knadler, Corey James
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The repair of double strand breaks (DSB) is a fundamental process that plays a key role in cell survival and evolution. DSB repair is required for cell survival, and a variety of pathways have evolved. Homologous recombination (HR) is a universal pathway of DSB repair found in nearly every species studied. HR encompasses several mechanisms, but key processes are conserved. The most fundamental of these activities are homology search and formation of heteroduplex DNA with double-stranded DNA (dsDNA) templates by means of the RecA family recombinases. This allows replication across the break site and subsequent repair of DSBs without mutation if an identical template dsDNA is used. Without identical template dsDNAs, HR can be a dangerous prospect leading to chromosomal rearrangements and gene conversions. As a result, two primary mechanisms have evolved to repair DSBs in the absence of dsDNA templates. The first is non-homologous end-joining, which uses end-binding, processing, and ligation proteins to repair broken ends with minor mutations. The second is a group of homology-based pathways that align broken dsDNAs on sequences repeated on either side of a break and delete the intervening sequence. In this document, new data is presented and examined regarding the structure-function- environment relationships of an archaeal recombinase, Sulfolobus solfataricus RadA (SsoRadA), and the proteins that modulate its activity. The ability of the organism S. solfataricus to perform end-joining was also examined for the first time. An overview of archaeal research and DSB repair across the three domains of life is provided in chapter one. In chapter two, the effects of divalent metal cofactors on SsoRadA recombinase function are examined, and it is revealed that SsoRadA has evolved to utilize divalent metals in an original way. Chapter three details, the first characterization of the SsoRal2 protein and its unusual action as an antagonistic regulator of SsoRadA activity. Chapter four details the ability of S. solfataricus to perform homology-based end-joining and investigates the availability of required homology throughout the S. solfataricus genome. In the final chapter, the implications of this work, and future areas of inquiry are discussed.