Dimerization through the RING-Finger Domain Attenuates Excision Activity of the piggyBac Transposase

The movement of the piggyBac transposon is mediated through its cognate transposase. The piggyBac transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac transposase. This attribute of the piggyBac transposase may serve to prevent excessive transposition of the piggyBac transposon that might be catastrophic for the host cell

The <i>N</i>²‑Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B‑DNA

<i>N</i>²-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model <i>N</i>²-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from <i>E. coli</i>. Introduction of fdG into a DNA duplex decreases the <i>T</i>_m by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W–C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct