Transcription Arrest by a G Quadruplex Forming-Trinucleotide Repeat Sequence from the Human c-myb Gene

Abstract

Non canonical DNA structures correspond to genomic regions particularly susceptible to genetic instability. The transcription process facilitates formation of these structures and plays a major role in generating the instability associated with these genomic sites. However, little is known about how non canonical structures are processed when encountered by an elongating RNA polymerase. Here we have studied the behavior of T7 RNA polymerase (T7RNAP) when encountering a G quadruplex forming-(GGA)(4) repeat located in the human c-myb proto-oncogene. To make direct correlations between formation of the structure and effects on transcription, we have taken advantage of the ability of the T7 polymerase to transcribe single-stranded substrates and of G4 DNA to form in single-stranded G-rich sequences in the presence of potassium ions. Under physiological KCl concentrations, we found that T7 RNAP transcription was arrested at two sites that mapped to the c-myb (GGA)(4) repeat sequence. The extent of arrest did not change with time, indicating that the c-myb repeat represented an absolute block and not a transient pause to T7 RNAP. Consistent with G4 DNA formation, arrest was not observed in the absence of KCl or in the presence of LiCl. Furthermore, mutations in the c-myb (GGA)(4) repeat, expected to prevent transition to G4, also eliminated the transcription block. We show T7 RNAP arrest at the c-myb repeat in double-stranded DNA under conditions mimicking the cellular concentration of biomolecules and potassium ions, suggesting that the G4 structure formed in the c-myb repeat may represent a transcription roadblock in vivo. Our results support a mechanism of transcription-coupled DNA repair initiated by arrest of transcription at G4 structures.

A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

“…G-quadruplex structure in a DNA strand impairs translocation of motor protein (29). In particular, it arrests RNA polymerase when it forms on the template strand (30). We modified the plasmid to have an SP6 promoter downstream of the T7 terminator and an SP6 terminator upstream of the T7 promoter (Figure 1C, scheme).…”

Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

“…It was previously reported that the formation of non-canonical structures, such as a G-quadruplex, reduce transcription efficiency because these structure induce pausing of the polymerase (42,43). For a template, we used a cholesterol-conjugated DNA with a G-quadruplex forming region near the 5′ end (Figure 6A).…”

“…Enthalpy–entropy compensation plot for the folding thermodynamics of ( A ) mcG n G-quadruplexes in the presence of POPC liposome and ( B ) G-quadruplexes in the absence of liposome (18,42,43). …”

Lesion Sensing and Decision Points in the DNA Damage Response

“…However, alternative DNA secondary structures, such as the G4 quadruplex, arrest transcription when they are formed in the non-transcribed strand. 12,13 Some types of damage, including the DNA adducts of aristocholic acid and the monofunctional adducts formed by irofulven (6-hydroxymethylacylfulvene), are transparent to recognition by the GGR pathway, but they can be sensitively detected by translocating RNAP, resulting in their efficient repair by TCR. 14,15 Of course, only the transcribed strands of expressed genes are being repaired in these cases; mutation-generating lesions persist in most of the genome.…”