• Dual- RNA Guided Editing of E.coli's DnaB Helicase Using the CRISPR/Cas9 System

      Jones, Preston Dimitri; College of Science and Mathematics; Spencer, Angie (2015-09-11)
      Precise manipulation of the genomic DNA is necessary for the characterization and identification of new genes and proteins. New technologies have enabled more facile and precise genomic engineering. Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein method affords precise manipulation of the genome. This system relies on a precise RNA guide (crRNA) that guides the cas9 nuclease to a specific location on the genomic, where it creates a double strand break (DSB). We can take advantage of this adaptive immunological advance and reprogram crRNAs to target whatever gene we like and introduce precise mutations to the genome by offering a designer template to introduce the changes and help the cell remain viable. This method is on the forefront of biochemistry and is being implemented in a numerous eukaryotic systems. However, it has yet to be fully utilized in bacterial genome editing. We are interested in exploring the bacterial helicase EcDnaB. The EcDnaB helicase has been hypothesized to unwind DNA in two fashions; both strands simultaneously or by unwinding one strand while excluding the other. Our lab postulates that there is a steric exclusion of one of the strands as it winds around the exterior of the helicase via electrostatic interactions. We used the CRISPR-Cas9 system to precisely mutate six loci on the EcDnaB gene, corresponding to amino acid residues on the external surface of the helicase, in order to better understand the mechanism of unwinding and to support our proposed method of unwinding. Begin Time: 37:59 End Time: 59:59
    • Investigating the role of Hob1 in Repairing Double Stranded DNA Breaks in the Fission Yeast, Schizosaccharomyces pombe

      Ozturk, Sarah; Abdulovic-Cui, Amy; College of Science and Mathematics (2015-02-20)
      Mutations in DNA induce many diseases, including cancer. The human protein, Binl, has anticancer properties and interacts with proteins involved in maintaining DNA stability. Work completed at the GRU Cancer Center has shown that Binl is specifically involved in the nonhomologous end-joining pathway (NHEJ), a pathway that repairs DNA breaks. To complement this work, we are investigating the role of Hobl, the homolog of Binl in fission yeast, in NHEJ, If Hobl functions in a similar manner to Binl, then removal of Hobl from yeast should decrease the cells ability to repair breaks in the DNA. We are testing this hypothesis using a genetic yeast transformation protocol that measures how efficient the yeast are at converting a linear piece of DNA into a repaired circular piece of DNA. Our initial data showed that yeast lacking the HOB1gene are 10 fold effective at repairing the linear DNA compared to wildtype yeast. These data were surprising as they contradict our hypothesis and the data collected in human cells that lack Binl. We are currently repeating the experiment to verify our results. Together our research supports a negative role for Hobl in repairing DNA double strand breaks in the fission yeast. Begin Time: 06:50 End Time: 28:24