Transposable element derived sequences comprise -40% of the human genome. Long Interspersed Elements (L1s or LINEs) comprise ∼17% of the genome and mobilize via an RNA intermediate by a process termed retrotransposition. L1 encodes two proteins (ORF1p and ORF2p), which are essential for mobility. ORF1p is an RNA binding protein, whereas ORF2p exhibits reverse transcriptase and endonuclease activities. L1 integration is thought to occur by target-site primed reverse transcription (TPRT). During TPRT, the L1 endonuclease cleaves the genomic DNA at the consensus sequence 5'TTTT/A (where the / indicates the cleavage site), exposing a 3' hydroxyl residue that serves as a primer for reverse transcription of the L1 RNA by the L1 reverse transcriptase. The nascent L1 cDNA then joins to genomic DNA generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3'poly(A) tail, and variable-length target site duplications (TSDs). An additional pathway for L1 insertion appears to act independently of endonuclease cleavage, but is dependent upon reverse transcriptase. Endonuclease-independent (ENi) retrotransposition occurs at near wild type levels in DNA-PKcs and XRCC4 deficient cell lines, two components utilized for the DNA repair pathway of non-homologous end joining (NHEJ). Analysis of the pre- and post-integration sites revealed that ENi retrotransposition results in unusual structures because the L1s integrate at atypical target sequences, are frequently truncated at both their 5' and 3' ends, and lack TSDs. We also detect ∼30% of ENi retrotransposition events insert in an orientation-specific manner adjacent to telomere repeats, in DNA-PKcs deficient cells which also display a telomere defect, whereas none were detected from XRCC4-deficient cells. Finally, we find L1 retrotransposition in NHEJ deficient cells only requires ORF1p and the RT domains, since mutations in the C-domain are also retrotransposition competent in NHEJ deficient cells and appear to integrate by a similar mechanism as ENi retrotransposition. In sum, L1 may integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.Ph.D.Biological SciencesGeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124894/2/3163893.pd
