REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application

<p>We have modified the combinatorial selection method Restriction Endonuclease Protection and Selection Assay (REPSA) to work in high temperature conditions for the discovery of new DNA-binding proteins in thermophiles (HT-REPSA). We utilized <em>Thermus thermophilus </em>(HB-8/ATCC 27634/DSM 579) as a test organism due to its amenable nature in a laboratory setting and current status as a model thermophilic organism. We used a TetR Family (TFR) transcription factor SbtR as the model protein for optimization of HT-REPSA protocols, as data had previously been obtained regarding SbtR physical characteristics and DNA-binding properties. REPSA was conducted until a cleavage resistant species arose after 7 rounds. Massively parallel sequencing of the selected DNAs and bioinformatics analysis yielded a consensus binding sequence of 5'-GA(t/c)TGACC(c/a)GC(t/g)GGTCA(g/a)TC, a 20base pair palindromic site comparable to that described in the literature. Taken together, our data provide a proof-of-concept that HT-REPSA can be successfully used to identify the preferred DNA-binding sequences of transcription factors from extreme thermophilic organisms.</p>

Name thesis:degree_name

Master of Science in Chemical Sciences (MSCB)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2015

Author dc:creator

• Beyer, Matthew

Contributors dc:contributor

• Dr. Michael Van Dyke
• Dr. Daniela Tapu
• Dr. Carol Chrestensen

• REPSA
• extremophile
• IISRE
• thermophilus
• thermophile
• Biochemistry

Repository record dc:identifier

https://digitalcommons.kennesaw.edu/mscs_etd/5

OAI identifier oai:identifier

oai:digitalcommons.kennesaw.edu:mscs_etd-1004