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The importance of L1 ORF2p cryptic sequence to ORF2p fragment-mediated cytotoxicity

Claiborne M Christian1, Kristine J Kines1, Victoria P Belancio1

  • 1Structural and Cellular Biology, Tulane University School of Medicine, Tulane Cancer Center, Tulane Center for Aging , New Orleans, LA, USA.

Mobile Genetic Elements
|September 2, 2016
PubMed

Insights

Long Interspersed Element 1 (LINE1) ORF2 protein fragments cause DNA damage. A newly identified Cryptic region (aa 270-274) in ORF2p influences fragment localization and cytotoxicity.

Area of Science:

  • Molecular biology
  • Genetics
  • Cell biology

Background:

  • Long Interspersed Element 1 (LINE1) retrotransposons are mobile genetic elements.
  • LINE1 ORF2 protein (ORF2p) possesses endonuclease (EN) activity, capable of inducing DNA double-strand breaks (DSB).
  • DSBs generated by ORF2p EN are potentially mutagenic, impacting genomic stability.

Purpose of the Study:

  • To investigate the role of specific amino acid sequences within ORF2p fragments on their subcellular localization and cytotoxicity.
  • To identify the functional significance of the newly discovered ORF2p Cryptic region (Cry).

Main Methods:

  • Expression of ORF2p fragments containing the endonuclease domain in mammalian cells.
  • Western blot analysis to detect protein presence and subcellular localization (nuclear vs. cytoplasmic).
  • Assessment of cytotoxicity associated with different ORF2p fragment constructs.

Main Results:

  • ORF2p fragments with the EN domain exhibit variable cytotoxicity in mammalian cells.
  • Including the C-terminal sequence to the EN domain reduced fragment cytotoxicity.
  • The C-terminal inclusion also increased nuclear localization of the EN-containing fragments.
  • Specific amino acids (aa 270-274) within the Cryptic region were identified as potentially crucial.

Conclusions:

  • The C-terminal region of ORF2p, including the Cryptic region, modulates the subcellular localization and cytotoxic effects of EN-containing fragments.
  • The identified amino acids (aa 270-274) in the Cryptic region are key determinants of these modulatory effects.
  • Understanding these regions is vital for comprehending LINE1-induced DNA damage and mutagenicity.
Keywords:
DNA damageL1ORF2endonucleaseretrotransposition

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