G-Quadruplex Forming Repeat Sequences In Bacterial Genomes

Rehm, Charlotte

G-Quadruplex Forming Repeat Sequences In Bacterial Genomes

Dateien

Rehm_0-299546.pdfGröße: 14.92 MBDownloads: 2365

Datum

2015

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Chemie

Publikationstyp

Dissertation

Publikationsstatus

Published

Zusammenfassung

Despite often being referred to as the inactive storage medium of genetic information DNA is of very dynamic and polymorphic nature adopting a variety of alternative secondary structures. In particular evidence for G-quadruplexes (GQPs), four-stranded helical complexes that are assembled from multiple stacked guanine tetrads, as important components in cellular processes has been increasing in recent years. These transiently formed alternative DNA structures have been shown to perform regulative roles in close to all integral biological processes such as recombination, replication, transcription and translation. In addition their polymorphic structure and high stability makes them attractive building blocks to be used in DNA nanoarchitectures and nanodevices.

In the first part of this thesis the GQP folding properties of the DNA sequence (G₄CT)₃G₄ were characterized. The G-rich sequence was recently identified as a potential quadruplex-forming sequence associated with the TprK antigen locus involved in antigenic variation in Treponema pallidum. Structural characterizations were carried out employing a combination of CD spectroscopy, EPR spectroscopy, analytical ultracentrifugation, NMR and EMSA. (G₄CT)₃G₄ displays remarkable properties such as a pronounced cation selectivity, folding kinetics with very high activation energies, and a continuous K⁺-dependent structural transition from an anti‐parallel topology at K⁺ concentrations as low as 0.5 mM to a parallel topology at 500 mM. We demonstrated that (G₄CT)₃G₄ is an intramolecular GQP in the antiparallel topology and tetramolecular in the parallel conformation. The effects of cation selectivity, quadruplex loop composition and length as well as G-tract length on GQP conversion were investigated.

Simple sequence repeats are highly abundant in the human genome and also ubiquitous in prokaryotes. Due to their repetitive nature they are prone to alternative secondary structure formation. Research on repetitive DNA motifs has so far been mostly focusing on eukaryotes, due to the involvement of trinucleotide expansions in human neurodegenerative diseases. In prokaryotes SSRs with unit sizes 1-5 nt were shown to be causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noted in bacteria, reports of studies of longer SSRs of 6-9 nt are rare. In particular the study of G-rich repeat sequences with the propensity to fold into GQP structures has been neglected.

The second part of this thesis focused on G-rich heptameric repeats of the type GGGAATC in Xanthomonas campestris pv. campestris ATCC 33913 and Xanthomonas axonopodis pv. citri str. 306. In addition GGGGA(C/T)T repeats in cyanobacterium Nostoc sp. strain PCC7120 were studied. The repeats were characterized with respect to their genomic distribution and location, length and sequence variability. In all three organisms repeats are spread ubiquitously all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed, however a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for GQP formation. Evidence for GQP formation of the consensus repeat sequences was found by the observation of characteristic spectral changes in CD spectroscopy. The G-rich repeats are preferably located between aligned ORFs (open reading frames) and are under-represented in coding regions or between divergent ORFs. The preference for the G-rich repeats to be located in close proximity up- and downstream of ORFs indicates a role as gene regulators. First studies toward elucidating a potential biological function of these sequence patterns were undertaken.

Taken together, both projects provide further insights into the dynamic and polymorphic nature of alternative secondary DNA structures. A connection between abundant G-rich repeat sequences and GQP forming sequences was made, topics that previously had been treated separately in the literature. Both studies provide starting points for further investigations, on the one hand for using GQPs as building blocks in nanotechnology and on the other hand to gain further insights into their proposed physiological roles as multifaceted regulatory DNA elements.

Fachgebiet (DDC)

540 Chemie

Schlagwörter

Non B-DNA, G-quadruplex, Simple Sequence Repeats, Bacteria

Zitieren

ISO 690

REHM, Charlotte, 2015. G-Quadruplex Forming Repeat Sequences In Bacterial Genomes [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Rehm2015GQuad-31737,
  year={2015},
  title={G-Quadruplex Forming Repeat Sequences In Bacterial Genomes},
  author={Rehm, Charlotte},
  address={Konstanz},
  school={Universität Konstanz}
}

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    <dcterms:abstract xml:lang="eng">Despite often being referred to as the inactive storage medium of genetic information DNA is of very dynamic and polymorphic nature adopting a variety of alternative secondary structures. In particular evidence for G-quadruplexes (GQPs), four-stranded helical complexes that are assembled from multiple stacked guanine tetrads, as important components in cellular processes has been increasing in recent years. These transiently formed alternative DNA structures have been shown to perform regulative roles in close to all integral biological processes such as recombination, replication, transcription and translation. In addition their polymorphic structure and high stability makes them attractive building blocks to be used in DNA nanoarchitectures and nanodevices.&lt;br /&gt;&lt;br /&gt;In the first part of this thesis the GQP folding properties of the DNA sequence (G&lt;sub&gt;4&lt;/sub&gt;CT)&lt;sub&gt;3&lt;/sub&gt;G&lt;sub&gt;4&lt;/sub&gt; were characterized. The G-rich sequence was recently identified as a potential quadruplex-forming sequence associated with the TprK antigen locus involved in antigenic variation in Treponema pallidum. Structural characterizations were carried out employing a combination of CD spectroscopy, EPR spectroscopy, analytical ultracentrifugation, NMR and EMSA. (G&lt;sub&gt;4&lt;/sub&gt;CT)&lt;sub&gt;3&lt;/sub&gt;G&lt;sub&gt;4&lt;/sub&gt; displays remarkable properties such as a pronounced cation selectivity, folding kinetics with very high activation energies, and a continuous K&lt;sup&gt;+&lt;/sup&gt;-dependent structural transition from an anti‐parallel topology at K&lt;sup&gt;+&lt;/sup&gt; concentrations as low as 0.5 mM to a parallel topology at 500 mM. We demonstrated that (G&lt;sub&gt;4&lt;/sub&gt;CT)&lt;sub&gt;3&lt;/sub&gt;G&lt;sub&gt;4&lt;/sub&gt; is an intramolecular GQP in the antiparallel topology and tetramolecular in the parallel conformation. The effects of cation selectivity, quadruplex loop composition and length as well as G-tract length on GQP conversion were investigated.&lt;br /&gt;&lt;br /&gt;Simple sequence repeats are highly abundant in the human genome and also ubiquitous in prokaryotes. Due to their repetitive nature they are prone to alternative secondary structure formation. Research on repetitive DNA motifs has so far been mostly focusing on eukaryotes, due to the involvement of trinucleotide expansions in human neurodegenerative diseases. In prokaryotes SSRs with unit sizes 1-5 nt were shown to be causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noted in bacteria, reports of studies of longer SSRs of 6-9 nt are rare. In particular the study of G-rich repeat sequences with the propensity to fold into GQP structures has been neglected.&lt;br /&gt;&lt;br /&gt;The second part of this thesis focused on G-rich heptameric repeats of the type GGGAATC in Xanthomonas campestris pv. campestris ATCC 33913 and Xanthomonas axonopodis pv. citri str. 306. In addition GGGGA(C/T)T repeats in cyanobacterium Nostoc sp. strain PCC7120 were studied. The repeats were characterized with respect to their genomic distribution and location, length and sequence variability. In all three organisms repeats are spread ubiquitously all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed, however a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for GQP formation. Evidence for GQP formation of the consensus repeat sequences was found by the observation of characteristic spectral changes in CD spectroscopy. The G-rich repeats are preferably located between aligned ORFs (open reading frames) and are under-represented in coding regions or between divergent ORFs. The preference for the G-rich repeats to be located in close proximity up- and downstream of ORFs indicates a role as gene regulators. First studies toward elucidating a potential biological function of these sequence patterns were undertaken.&lt;br /&gt;&lt;br /&gt;Taken together, both projects provide further insights into the dynamic and polymorphic nature of alternative secondary DNA structures. A connection between abundant G-rich repeat sequences and GQP forming sequences was made, topics that previously had been treated separately in the literature. Both studies provide starting points for further investigations, on the one hand for using GQPs as building blocks in nanotechnology and on the other hand to gain further insights into their proposed physiological roles as multifaceted regulatory DNA elements.</dcterms:abstract>
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Prüfungsdatum der Dissertation

March 24, 2015

Hochschulschriftenvermerk

Konstanz, Univ., Diss., 2015

Universitätsbibliographie

Ja