Mechanistically, Topo II functions to alter DNA topology by catalyzing the ATP-dependent passage of one DNA double helix through another by breaking and religating one of the DNA strands while transporting the second [26], [29], [30], [31].
pCZ486 contains a deletion only in the drsh-1 coding region and was constructed by digesting pCZ483 with SphI and religating.
To generate pBac17, pVT1 DNA was digested by NotI and religated between the two NotI sites of pBeloBac11 [33].
The DNA encoding eGFP was removed from pAct5eGFP by cutting with BamHI and XbaI, blunting using Klenow polymerase and religating to yield pAct5c.
Empty vector control was obtained by cleaving the IFI16 insert out of CMV-SPORT6 using NotI/ SalI and religating the vector.
The BP donor was constructed by digesting the 7.5-kb donor with BssHII and PmlI, then blunting and religating as above, creating a 1089-bp dendingn ending 825 bp upstream from the target site.
The BX donor was constructed by digesting the 7.5-kb donor with XbaI and BspDI, then blunting and religating as above, creating a 1232-bp deletion beginning at the target site.
The SA donor was constructed by digesting the 7.5-kb donor with AflII and SgrAI and blunting and religating as above, creating a 1307-bp deletion beginning 699 bp upstream and ending 608 bp downstream of the target site.
NMHCIIA-ΔC-ter was generated by digesting pTRE GFP NMHCIIA with EcoRI and SpeI, blunting with Klenow and religating, thus creating a truncated fusion protein lacking the last 620 aa of MNHCIIA.
pGEM-uidA-BAR was created by inserting the NotI cassette of pFLP-SWITCH-BAR into the NotI site of a pGEM T-easy (Promega, Wisconsin, USA) derivative in which the SacI site in the multiple cloning site was removed by digesting with SacI, using T4 DNA Polymerase to make blunt ends, and religating the plasmid backbone.
