Analysis of DNA transposition by DNA transposases in human cells

Abstract

This manuscript discusses the recent report "Cognate restriction of transposition by piggyBac-like proteins" in Nucleic Acids Research by Beckermann et al and related recent publications about the inability to detect DNA transposition activity of human domesticated DNA transposase PGBD5. Measuring DNA transposition activity of transposases in human cells, where these enzymes can act on endogenous genomic substrates and induce DNA damage, is complicated by these and other cellular responses. Possible reasons for the discordant findings of Beckermann et al with prior independent reports of PGBD5 DNA transposition by Helou et al and Henssen et al and specific details of experimental methods in human cells are presented. In particular, by using independent experiments that reproduce PGBD5-mediated genomic integration, we demonstrate how supraphysiologic and ectopic overexpression of PGBD5 can cause DNA damage and cell death, and artifactual loss of apparent activity in clonogenic transposition reporter assays. While PGBD5 can support apparent DNA transposition, its cellular activity predominantly involves double-strand DNA breaks, deletions and other DNA rearrangements. We discuss the implications of this phenomenon for the interpretation of experimental assays and activities of domesticated DNA transposases.

Childhood cancer mutagenesis caused by transposase-derived PGBD5

“…This is an important direction of future work because two recent publications reported an inability to detect DNA transposition of PGBD5 in cellular assays (43,44). While PGBD5 can support genomic DNA integration in cells, its cellular activity predominantly involves double-strand DNA breaks, deletions, and other DNA rearrangements, with relatively few precise transposon-specific excisions and insertions (8). Thus, human PGBD5 is divergent from "cut-and-paste" transposases such as Trichoplusia ni piggyBac, but rather should be considered a transposase-derived protein with domesticated genomic integration and DNA rearrangement activities in human cells.…”

“…Although PGBD5 can support genomic DNA integration in cells, its cellular activity predominantly involves double-strand DNA breaks, deletions, and other DNA rearrangements. While the specific enzymatic mechanisms of PGBD5-induced genome rearrangements need to be defined, PGBD5 can mediate rearrangements of both heterologous transposon substrates as well as sequence elements in the human and mouse genomes (3,4), as validated by Helou et al (6,7) and recently confirmed by Bigot et al (8).…”