Date on Honors Thesis

Fall 11-12-2020





Examining Committee Member

Christopher Lennon, Ph.D., Advisor

Examining Committee Member

Gary ZeRuth, Ph.D., Committee Member

Examining Committee Member

Terry Derting, Ph.D., Committee Member


Inteins are protein segments interrupting polypeptides with the unique ability to excise from the host protein and link flanking protein fragments (exteins) to form a functional protein. Many of these proteins are involved in crucial processes such as DNA replication, DNA recombination, and DNA repair. The functionality of these elements in nature and applications in biotechnology necessitates the study of inteins. Pyrococcus horikoshii (Pho) provides an array of inteins to study, with a total of nine different proteins possessing inteins. Among the nine total inteins, the DNA repair and recombination protein RadA gained interest due to the speed and efficiency of splicing when flanked by non-native exteins. Curiously, RadA splices poorly within the native exteins in the absence of catalysts due to intein-extein interactions. Single-stranded DNA (ssDNA), which binds to RadA on the c-extein, vastly improves the speed and accuracy of splicing. ssDNA is not only a substrate of the RadA protein but also a signal indicating DNA damage and the need for recombinase activity. Given the observation that intein-extein interactions form to block splicing, we wondered how alternative intein insertion sites within the host protein would influence splicing. Observation and comparison of splicing speed and efficiency in natural and variant RadA proteins may help us further understand the phenomena of intein splicing, explain intein-extein relationships, and give insight on practical applications.

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