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Properties of “sticky DNA” formed by GAA repeats

Properties of “sticky DNA” formed by GAA repeats
Properties of “sticky DNA” formed by GAA repeats
Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by long repeats of the trinucleotide GAA.TTC in the first intron of the frataxin (FXN) gene. This expanded repeat results in lower than normal levels of mature FXN mRNA and thus reduced levels of frataxin protein, the FXN gene product. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have from 70 to more than 1000 GAA triplets. The frataxin protein plays a role in assembly of iron-sulphur clusters which are needed for energy production. As GAA.TTC repeats contain only purine bases (Pu) on one strand and pyrimidine bases (Py) on the complementary DNA strand, they can form intramolecular triplexes under superhelical stress. “Sticky DNA” is a poorly characterised DNA structure that is generated by the association of two long stretches of the trinucleotide GAA.TTC sequence (>59 repeats), which are found in FRDA. The formation of this structure is dependent on the length and orientation of the repeats, negative supercoiling and the presence of divalent metal ions. Although sticky DNA was discovered over a decade ago, its exact structure remains unknown. Therefore, the principle aim of this thesis was to examine and characterize the formation of non-B-DNA structures in the FRDA-associated GAA.TTC repeats that were cloned in suitable vectors. Various lengths of GAA.TTC repeats (up to 85 repeats) were cloned into two sites in both pUC18 and pGL3 plasmids, either singly or in combination. The clones in pUC18 were used to assess the formation of unusual DNA structures (H-DNA or sticky DNA), through the use of the chemical probes diethylpyrocarbonate (DEPC) and potassium permanganate (KMnO4), and the single-strand specific endonuclease S1. These react with the regions of single stranded DNA that are generated within the non-B DNA structures. The use of DEPC and KMnO4 together provided us a complete analysis of both the GAA- and TTC-containing DNA strands. DEPC and KMnO4 revealed strong evidence for H-DNA formation in short (GAA.TTC)n sequences (n ≤ 29) and some evidence in longer inserts. However, there was no evidence for ‘sticky DNA’ formation within our plasmids. High resolution probing with S1 nuclease confirmed the DEPC and KMnO4 data for detecting H-DNA in short inserts, but failed to detect any unique structural perturbations within the longer repeats. However, when S1 nuclease was used to map the single strand regions that accompany H-DNA formation, S1 sensitive sites were apparent for both short and long GAA.TTC repeats, indicating the presence of DNA secondary structure within this region, which was only present in the supercoiled species. The effects of GAA.TTC repeats on the structure of the plasmids were also examined by observing their electrophoretic mobility in agarose gels. The results showed that plasmids containing long GAA-repeats exhibited some unusual properties and were prone to form multimeric structures (dimers, trimers etc.). By using limited restriction digestions, we confirmed that the low mobility plasmid species is a dimer that consists of two monomer plasmids covalently ligated together. However, we failed to detect any ‘sticky DNA’ conformation in any of the cloned pUC18 plasmids, even those containing two tracts of long repeats. Finally, GAA-inserts in pGL3 were used to assess the effect of these sequences on transcription of the adjacent luciferase gene. These results reveal that a decrease in gene expression level from the vectors that carry short or long repeats at one or two positions in the luciferase reporter vector.
University of Southampton
Alsulami, Mohammed Sulaiman
58248a23-ac4b-43ca-87d8-78a923b2fc8b
Alsulami, Mohammed Sulaiman
58248a23-ac4b-43ca-87d8-78a923b2fc8b
Fox, Keith
9da5debc-4e45-473e-ab8c-550d1104659f

Alsulami, Mohammed Sulaiman (2016) Properties of “sticky DNA” formed by GAA repeats. University of Southampton, Doctoral Thesis, 214pp.

Record type: Thesis (Doctoral)

Abstract

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by long repeats of the trinucleotide GAA.TTC in the first intron of the frataxin (FXN) gene. This expanded repeat results in lower than normal levels of mature FXN mRNA and thus reduced levels of frataxin protein, the FXN gene product. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have from 70 to more than 1000 GAA triplets. The frataxin protein plays a role in assembly of iron-sulphur clusters which are needed for energy production. As GAA.TTC repeats contain only purine bases (Pu) on one strand and pyrimidine bases (Py) on the complementary DNA strand, they can form intramolecular triplexes under superhelical stress. “Sticky DNA” is a poorly characterised DNA structure that is generated by the association of two long stretches of the trinucleotide GAA.TTC sequence (>59 repeats), which are found in FRDA. The formation of this structure is dependent on the length and orientation of the repeats, negative supercoiling and the presence of divalent metal ions. Although sticky DNA was discovered over a decade ago, its exact structure remains unknown. Therefore, the principle aim of this thesis was to examine and characterize the formation of non-B-DNA structures in the FRDA-associated GAA.TTC repeats that were cloned in suitable vectors. Various lengths of GAA.TTC repeats (up to 85 repeats) were cloned into two sites in both pUC18 and pGL3 plasmids, either singly or in combination. The clones in pUC18 were used to assess the formation of unusual DNA structures (H-DNA or sticky DNA), through the use of the chemical probes diethylpyrocarbonate (DEPC) and potassium permanganate (KMnO4), and the single-strand specific endonuclease S1. These react with the regions of single stranded DNA that are generated within the non-B DNA structures. The use of DEPC and KMnO4 together provided us a complete analysis of both the GAA- and TTC-containing DNA strands. DEPC and KMnO4 revealed strong evidence for H-DNA formation in short (GAA.TTC)n sequences (n ≤ 29) and some evidence in longer inserts. However, there was no evidence for ‘sticky DNA’ formation within our plasmids. High resolution probing with S1 nuclease confirmed the DEPC and KMnO4 data for detecting H-DNA in short inserts, but failed to detect any unique structural perturbations within the longer repeats. However, when S1 nuclease was used to map the single strand regions that accompany H-DNA formation, S1 sensitive sites were apparent for both short and long GAA.TTC repeats, indicating the presence of DNA secondary structure within this region, which was only present in the supercoiled species. The effects of GAA.TTC repeats on the structure of the plasmids were also examined by observing their electrophoretic mobility in agarose gels. The results showed that plasmids containing long GAA-repeats exhibited some unusual properties and were prone to form multimeric structures (dimers, trimers etc.). By using limited restriction digestions, we confirmed that the low mobility plasmid species is a dimer that consists of two monomer plasmids covalently ligated together. However, we failed to detect any ‘sticky DNA’ conformation in any of the cloned pUC18 plasmids, even those containing two tracts of long repeats. Finally, GAA-inserts in pGL3 were used to assess the effect of these sequences on transcription of the adjacent luciferase gene. These results reveal that a decrease in gene expression level from the vectors that carry short or long repeats at one or two positions in the luciferase reporter vector.

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Published date: August 2016

Identifiers

Local EPrints ID: 413592
URI: https://eprints.soton.ac.uk/id/eprint/413592
PURE UUID: e00962c2-1a2a-4da3-9487-f864bb486cd8
ORCID for Keith Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 29 Aug 2017 16:30
Last modified: 14 Mar 2019 01:56

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Contributors

Author: Mohammed Sulaiman Alsulami
Thesis advisor: Keith Fox ORCID iD

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