Date of Award

2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Chair

Joseph Ng

Committee Member

Deborah M. Moriarity

Committee Member

Joseph G. Leahy

Subject(s)

DNA ligases., Phosphodiesterases., DNA polymerases.

Abstract

DNA ligases are highly conserved in their primary sequence across the three domains of life and are important in DNA replication, recombination, and repair. The enzyme functions in an AMP-dependent reaction to close nicks in the DNA phosphodiester backbone. In this study, the hyperthermophilic DNA ligase Tthlig from the archaeon Thermococcus thioreducens is characterized. Tthlig is a recombinant protein cloned from genomic DNA with a sequence of 559 amino acid residues. Phylogenetic analysis placed Tthlig in a tight cluster with organisms of the order Thermococcales. Amino acid residue sequence analysis showed significant sequence conservation with other DNA ligases. The structure and domain assignment was predicted against the crystallographic structure of Thermococcus sp. 1519. Tthlig was purified to over 90% homogeneity with one heat selection and 3 chromatographic steps. The overall yield for an established purification protocol was 1.2 mg per gram of wet cell mass. The enzymatic activity of Tthlig was characterized at 750 C with nicked pUC18 as substrate and ATP as the source of AMP. The ligase was found to be most active in a 20mM Na-phosphate buffer, pH 7.0, 100mM KCl and 10mM MgCl2. Tthlig stability and activity at high temperatures qualifies it as a strong candidate in diagnostic techniques to detect point mutations associated with specific diseases.

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