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

真核生物中的DNA修饰是重要的表观遗传标记，在调节基因组活性和稳定性方面发挥着核心作用。虽然大多数研究都是针对识别和翻译这种表观遗传信息的蛋白质，但我们想要研究这些DNA修饰如何直接和间接地影响DNA双螺旋的稳定性，以及如何影响基本的细胞过程，如转录和复制。DNA双螺旋的局部失稳可能会影响碱基翻转等过程，如修改或修复碱基，在转录启动过程中打开复合体形成，或调节转录速度，从而影响相关过程，如RNA加工或RNA引导的表观遗传修饰。我们将在体外直接测量已知的DNA修饰对DNA双螺旋稳定性以及对DNA/RNA聚合酶和DNA解旋酶的影响。然后，我们将使用遗传方法在体细胞和同基因胚胎干细胞中增加或减少碱基修饰的水平，并将在体内测量核糖核苷酸和脱氧核糖核苷酸聚合的速率。我们还将通过将DNA聚合从细胞复制体内的DNA解旋酶活性中分离出来，来测量体内DNA解旋酶的速率。

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.