Analysis Of The Line-1 Orf2 Protein Using An Evolutionarily-informed Genetic Approach
Long Interspersed Element 1(LINE1 or L1), along with the parasitic Short Interspersed Element (SINE), are the only two currently active retrotransposons in the human genome. These genetic elements are capable of causing DNA damage through their mobilization and the enzymatic activities of the L1-encoded Open Reading Frame 2 (ORF2) protein (ORF2p). The L1 ORF2p contains four annotated domains important to retrotransposition. These include the endonuclease (EN) and reverse transcriptase (RT) domains, as well as the Z domain and the cysteine rich domain (Cys). While much is known about the enzymatic activities of the EN and RT domains, and individual amino acids important to retrotransposition have been identified in the Z and Cys domains, more than 50% of the 150kDa ORF2p amino acid sequence serves no known function. I hypothesized that the unannotated areas of the ORF2p, specifically the sequence C-terminal to the EN domain and N-terminal to the Z domain as well as the sequence C-terminal to the Cys domain, contained amino acids important to the retrotransposition process. Specifically, I hypothesized that they contained amino acids involved in the activity of the EN domain, RT domain, or interaction with the L1 ORF1p. To test this hypothesis, I developed a technique termed Bipartile Alu Retrotransposition (BAR) that utilized EN and RT-containing ORF2 fragments combined with the Alu retrotransposition reporter construct. This system allowed me to define a new ORF2p region, which I termed Cryptic. This region contains an essential WD pair important for cDNA syntheses by the ORF2p. This WD pair is also involved in the regulation of the EN domain activity. I also discovered a putative PCNA binding domain that is essential for retrotransposition. Additionally, I identified the region in Cryptic that is involved in the previously reported differences in subcellular localization and cytotoxic potential of EN-containing ORF2p fragments. Using truncated ORF2p fragments generated for use in BAR, I also made several discoveries concerning the extreme C-terminus of the ORF2p. Notably, I discovered that the extreme C-terminal end of the ORF2p is dispensable for retrotransposition. I also identified a human-specific Y residue that is important for Alu retrotransposition driven by the ORF2p.