The linker region of LINEs modulates DNA cleavage and DNA polymerization

Anal Biochem. 2020 Aug 15;603:113809. doi: 10.1016/j.ab.2020.113809. Epub 2020 Jun 6.

Abstract

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.

Keywords: DNA endonuclease; LINE; Non-LTR retrotransposon; Reverse transcriptase; Target primed reverse transcription; Transposable element.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Conserved Sequence
  • DNA / biosynthesis
  • DNA / chemistry*
  • DNA / metabolism*
  • DNA Cleavage
  • Deoxyribonuclease I / metabolism
  • Insect Proteins
  • Long Interspersed Nucleotide Elements / physiology*
  • Point Mutation
  • Polymerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA-Directed DNA Polymerase / metabolism

Substances

  • Insect Proteins
  • DNA
  • R2 reverse transcriptase, Bombyx mori
  • RNA-Directed DNA Polymerase
  • Deoxyribonuclease I