REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application
Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Michael Van Dyke
Dr. Daniela Tapu
Dr. Carol Chrestensen
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 Thermus thermophilus (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.