Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Skyler Taylor - Xavier University of Louisiana
Co-Author(s): Derian Pugh, Xavier University of Louisiana, New Orleans, LA; Brittany Russell, Xavier University of Louisiana, New Orleans, LA; Cecily DeFreece, Xavier University of Louisiana, New Orleans, LA
Abstract Introduction Mobile elements are ubiquitous in the human genome. The mobile elements Long Interspersed Element-1 (L1) and Short Interspersed Element (SINE) Alu are retrotransposons that comprise approximately 30% of the human genome. Both mobile elements move in the genome using a copy and paste mechanism termed retrotransposition. The mobile element Alu does not encode any proteins and requires L1 for retrotransposition. L1 is comprised of two open reading frames (ORF), ORF1 and ORF2. The ORF1 protein of L1 encodes an RNA binding protein that has molecular chaperone activity. The ORF2 protein encodes an endonuclease (EN), and reverse transcriptase (RT); both enzymes are required for retrotransposition. Previous research has shown that the heavy metal lead prevents retrotransposition of the mobile element Alu. We hypothesize that lead is preventing the activity of the L1 EN and as a result inhibiting Alu retrotransposition. We will perform in vitro experiments using purified L1 EN to demonstrate the effect of lead on L1 EN activity. Methods Purification – The L1 EN was cloned into a pET15b vector to generate an amino terminal HIS-tagged protein upon expression. The expression was induced with 1mM IPTG at 23℃ overnight. L1 EN His-tagged protein was purified using Nickel affinity chromatography. Cleavage assay- This assay was performed using 200mM purified L1 EN, 100mM duplexed fluorescently tagged oligonucleotide with L1 EN target site. The control assay buffer is 20mM Hepes (pH 6.5), 150 mM NaCl, 1 mM Mg, 0.1 mg/mL BSA. The buffer for lead contained 20 mM Hepes (pH 6.5), 150 mM NaCl, 1 mM Pb, 0.1 mg/mL BSA. Assays were carried out at 37℃ for 30 minutes. The reaction were stopped by quenching on ice and with the addition of a stop solution. The collected samples were run on 18% denaturing acrylamide gels and the fluorescence intensity was measured using typhoon trio+ and products quantitated using Image Quant TL. Results and Conclusions We observed an approximately 50% reduction in L1 EN enzymatic activity when 1 mM of lead was used in the assay as compared to 1 mM of magnesium. The inhibition of the cleavage is either due to the prevention of L1 EN from binding, or solely related to the inability of the L1 EN to cleave after exposed to the lead. Future Directions We intend to determine if the inhibition of L1 EN activity by lead occurs in a dose dependent manner. We will also test if the effect can be rescued by addition of increasing amounts of magnesium. Furthermore, we intend to determine if lead is preventing binding of the oligonucleotide substrate to the L1 EN or is preventing cleavage of the substrate. These experiments will indicate which step in the enzymatic mechanism is being affected by lead. Overall these studies will demonstrate whether the reduction in Alu retrotransposition previously observed is a result of the inhibition of L1 EN activity.Abstract.2.docx
Funder Acknowledgement(s): LS-Lamp National Institute of General Medical Sciences (NGIMS) 8P20GM103424
Faculty Advisor: Dr. Cecily DeFreece, firstname.lastname@example.org
Role: I tested the effects of metals on the pH of the buffer to show that the loss of enzyme activity was not due to the change in pH. I also performed the binding assay to determine whether lead (Pb2+) affected the ability of L1 EN to bind to the oligonucleotide.