Biophysikalische Charakterisierung und biochemische Steuerung von Methyltransferase-DNA-Wechselwirkungen

This work explores the use of biophysical methods for the characterisation of DNA methyltransferase (MTase) interactions with DNA and their methodical direction by means of biochemical approaches. Taking the MTase M.TaqI as a paradigm, the kinetic analysis of these interactions is performed by stopped flow measurements with detection of the UV absorption. Compared to the more elaborate monitoring of synthetic fluorescent labels this method enables the investigation of the unmodified natural system. Due to the numerous chromophores it may also allow the detection of hitherto unknown kinetic steps, which is highlighted in the present work. Another project deals with diverse strategies for the fluourescent labelling of short duplex-DNA with regard to the analysis of M.TaqI-DNA-interactions using fluorescence anisotropy. In this study, a topology with forced intercalation of the fluorescent probe emerged as the most suited alternative. It shows a huge fluorescence anisotropy increase and no significant fluorescence intensity change upon M.TaqI binding. An analogously probed duplex-DNA was taken to determine a reliable dissociation constant for the M.TaqI-DNA complex which was found to be Kd = 0.34 nM. Because of the great complex stability, former studies with 2 aminopurine labeled DNA led to rather diverse parameters. In contrast to forced intercalation of the fluorescent probe, topologies with a rather mobile fluorescent probe at the 5’-end of the duplex-DNA turned out to be prone to unwanted interactions with the enzyme or to a marginal fluorescence anisotropy increase. The second part of this work consists of four approaches to biochemically direct the MTase-DNA interaction in different ways. One strategy was to take advantage of the known aziridine cofactor analogue Az which is covalently coupled to the target DNA by several MTases. The formed complexes represent bisubstrate analogues and are extremely stable. In the case of M.TaqI and M.HhaI their dissociation rates are 5,000 to 10,000 times slower than the MTase complexes with the natural methylation products. Accordingly, their thermodynamic dissociation constants are also several magnitudes lower than those of the natural product complexes which render these bisubstrate analogues to be promising candidates for DNA-MTase inhibitors. The replacement of the natural target base partner in the M.TaqI recognition sequence by hydrophobic base surrogates also led to a dramatic increase in the DNA binding affinity of M.TaqI of up to three magnitudes. The dissociation constants of multiple compounds were measured by the use of a competitive binding assay with pyrene-labelled duplex-DNA. The gathered data give insight into  stacking interactions in enzyme-DNA complexes and can be utilized for the investigation of DNA binding mechanisms of other enzymes. In a competitive fluorescence binding assay with 2-aminopurine-labelled DNA, it was demonstrated that distance-constrained macrocyclic bisintercalators represent a tool to biochemically influence the binding of M.TaqI and presumably of repair enzymes to mismatched DNA. In addition, the mismatch binding specificities of several bisintercalators were characterised in good agreement by several independent bioanalytical methods. These results suggest that the crucial detection of point mutations within the genome can be achieved with this sort of molecules, but it would make the conjugation of the bisintercalators to robust reporting groups, e.g. fluorophors, necessary. In the last chapter, the DNA-binding properties of an MTase which is covalently linked to a triplehelix-forming oligonucleotide (TFO) were investigated. This conjugate has been synthesised in previous works and exhibits dramatically increased sequence specificity compared to the unmodified MTase. A very sensitive and straightforward fluorescence assay was developed within the scope of the present work that enables the observation of the conjugated TFO binding to duplex-DNA. The assay allows for measurements at low nanomolar concentrations, e.g. real-time monitoring of the TFO binding to plasmid-DNA or the estimation of the dissociation constant of the formed triplehelix.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2009

Author dc:creator

• Bahr, Matthias

Contributors dc:contributor

• Weinhold, Elmar

• info:eu-repo/classification/ddc/540
• DNS-Methyltransferase
• DNS
• Enzym
• Biochemische Analyse
• Fluoreszenzspektroskopie
• Chemie
• Biophysikalische Analyse
• DNA
• enzyme
• DNA-methyltransferase
• fluorescence spectroscopy
• bioanalytics

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113622%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51319