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. 2012 Sep 8;40(20):10334–10344. doi: 10.1093/nar/gks802

Figure 5.

Figure 5.

Hybrid G-quadruplexes form during transcription and are dependent on CSB II. (A) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. (B) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. (C) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.