Impact of DNA base modifications on the double helix stability and genome activity

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DNA modifications in eukaryotic organisms are important epigenetic marks and play a central role in regulation of genome activity and stability. While most research is geared towards proteins recognizing and translating this epigenetic information, we want to investigate how these DNA modifications directly and indirectly affect the stability of the DNA double helix and how that affects basic cellular processes like transcription and replication.Local destabilization of the DNA double helix could affect processes like base flipping to modify or repair bases, open complex formation during transcription initiation or modulate the speed of transcription and thereby affect associated processes like RNA processing or RNA guided epigenetic modifications. We will directly measure the effect of known DNA modifications on DNA double helix stability and on DNA/RNA polymerases and DNA helicases in vitro. We will then increase or decrease the level of base modifications using genetic approaches in somatic as well as isogenic embryonic stem cells and will measure rate of ribonucleotide as well as desoxyribonucleotide polymerization in vivo. We will also measure the rate of DNA unwinding by DNA helicase in vivo by uncoupling the DNA polymerization from the DNA helicase activities within the cellular replisome.