Biochemical analysis of chromatin targeting by Tn5 transposase

Tn5 transposase is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes, but its DNA target site in chromatin has not been understood. In the present study, the well-positioned dinucleosomes were reconstituted, and the Tn5 transposase target sites were mapped in the dinucleosomes in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA region within the nucleosome, if the linker DNA exists between two nucleosomes. This specific DNA targeting by Tn5 did not depend on the linker DNA length and DNA sequence. Tn5 transposase becomes to target the middle of the linker DNA, in addition to the entry-exit site of the nucleosome, if the linker DNA length extends to 30 base pairs. These in vitro data provide direct evidence for the Tn5 target sites in the nucleosome, resulting important information for interpretation of the Tn5-transposase-based genomics methods, which have been interpreted as linker or nucleosome-free DNA regions in genomes. Overall design: To understand the Tn5 transposase target site in nucleosomal DNA, four dinucleosomes with different linker DNA lengths and DNA sequences were reconstituted as substrates, and the Tn5 transposase reactions were performed in vitro.