The linker region of LINEs modulates DNA cleavage and DNA polymerization

• Published in: Analytical biochemistry 2020-08, Vol.603, p.113809-113809, Article 113809
• Main Authors: Pradhan, Monika, Govindaraju, Aruna, Jagdish, Athena, Christensen, Shawn M.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Binding Sites; Conserved Sequence; Deoxyribonuclease I - metabolism; DNA - biosynthesis; DNA - chemistry; DNA - metabolism; DNA Cleavage; DNA endonuclease; Insect Proteins; LINE; Long Interspersed Nucleotide Elements - physiology; Non-LTR retrotransposon; Point Mutation; Polymerization; Protein Structure, Secondary; Protein Structure, Tertiary; Reverse transcriptase; RNA-Directed DNA Polymerase - metabolism; Target primed reverse transcription; Transposable element
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113809

Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease. [Display omitted] •Linker region of RLE LINEs modulates first and second-strand DNA cleavage.•Linker region of RLE LINEs modulates first and second-strand DNA synthesis.•Linker region is a nucleic-acid/protein conformational switching platform.•Disruption of the linker leads to promiscuous DNA cleavage and inability to form RNP-DNA complexes.