GTMB 7 - Gene Therapy & Molecular Biology

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Buldak and Mirkin: Hypersensitivity of the d(GA) n •d(TC) n dependent on lactose repressorFigure 2. Summary of CAA modifications observed between positions -70 and +5 relative to the TSS. Modified cytosine and/or adenineresidues shown in red occurred only in the absence of IPTG. Modifications shown in green occurred only in the presence of IPTG.Modifications shown in turquoise occurred in both states of promoter induction.These data, therefore, rule out the possibility that themodification of the repeat results from the deformation atthe promoter region.Though modification of the d(GA) n •d(TC) n insert does notseem to be directly linked to the trc promoter activity, it isdowngraded in the presence of IPTG. Since the onlyknown effect of IPTG is to block DNA-binding activity ofthe lactose repressor, we looked at the role of the lactoserepressor in the d(GA) n •d(TC) n insert modification. Tothis end, we first deleted lacI q gene from our test plasmidand transformed the resultant construct into the lacI -background. The data on chemical probing in vivopresented in Figure 5 show that there are no modificationsof the d(GA) n •d(TC) n insert in this setting. At the sametime, the modifications within the promoter region wereidentical to those seen in previous experiments in thepresence of IPTG, showing that trc promoter was in aconstantly induced state. We conclude, therefore, thatfunctional lactose repressor is necessary for a structuralalteration of the d(GA) n •d(TC) n repeat leading to itschemical hyperreactivity in vivo.Lactose repressor is known to bind to at least twooperator sites in our plasmid. The next question istherefore, whether repressor-operator binding is essentialfor the repeat's modification. To answer this question, wedeleted the operator and pseudo-operator, eitherindividually or together. When the primary operator (O1)was deleted, the modification of the d(GA) n •d(TC) n repeatwas similar to that observed in the presence of the normaloperator site (Figure 6). The modification of the promoterregion, as one would expect, became equivalent to that ofthe induced wild-type promoter even in the absence ofIPTG. When the O3 pseudo-operator was deletedindividually, leaving O1 intact, modifications of both theinsert and of the promoter regions (Figure 7A) wereidentical to those seen in the original plasmid assummarized in Figure 2. Furthermore, when both operatorsites were deleted (ΔO1-ΔO3 construct), the results ofmodification were identical to those seen in the case of theΔO1 (Figure 7B). We conclude, therefore, that lactoserepressor per se, rather than its binding to the operatorsites, is necessary for the d(GA) n •d(TC) n hyperreactivity.Figure 3. CAA modification in vivo of the non-transcribedstrand of pTrcCat/Pst. G, C, Maxam-Gilbert sequencing reactionsof plasmid DNA in vitro. IPTG - and + indicate C reactions for invivo CAA-modified plasmid DNAs. Arrows indicate modifiedadenine and cytosine bases.IV. DiscussionWe found that a d(GA) n •d(TC) n repeat does undergostructural changes, accompanied by chemical modification294
Gene Therapy and Molecular Biology Vol 7, page 295Figure 5. CAA modification in vivo of the polypurine strand of ad(GA) 37 •d(TC) 37 repeat, inserted 50 bp upstream of the trc TSSin pTrcCat/Pst-GA37ΔlacI q . G, C, Maxam-Gilbert sequencingreactions of plasmid DNA in vitro. IPTG - and +, C reactions forplasmid DNAs that were CAA-modified in vivo in the absence orpresence of IPTG, respectively. The left panel shows thed(GA) 37 •(d(TC) 37 repeat with the promoter, while right panelonly the repeat. Arrows indicate modified adenine and cytosineresidues.Figure 4. CAA modification in vivo of the polypurine strand of ad(GA) 37 •d(TC) 37 repeat at position -180 relative to the TSS inpTrcCat/Cla-GA37. G, C, Maxam-Gilbert sequencing reactionsof plasmid DNA in vitro. IPTG - and + indicate CAAmodifications in vivo in the absence or presence of IPTG,respectively. IPTG 2' is a control in which the plasmid DNA wasCAA-modified in vivo for two minutes, instead of the normal 20minutes. Arrows indicate modified adenine residues.of the 3'-part of the polypurine strand, when situated in theupstream promoter area. Contrary to expectations, basedon previous studies by us and others, this modificationpattern is not immediately consistent with previouslydescribed types of non-canonical structures adopted bythis repeat.Based on our previous studies (Dayn et al, 1992;Krasilnikov et al, 1999), we assumed that transcriptionalsupercoiling would stimulate structural transitions withinthe d(GA) n •d(TC) n repeat.295
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GTMB 7 - Gene Therapy & Molecular Biology

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