Deciphering the molecular mechanisms of DNA replication and repair – integration of computational and experimental approaches

Deciphering the molecular mechanisms of DNA replication and repair – integration of computational and experimental approaches

Internship Description

Genomic DNA is under constant assault by environmental factors that introduce variety of DNA lesions. The cell evolved several DNA repair and recombination mechanisms to remove these damages and ensure the integrity of the genomic DNA. These mechanisms use DNA structures that deviate from the heritable duplex DNA (flaps, nicks, gaps, bubbles and four-ways Holliday junctions) as common pathway intermediates. However, these structures are extremely toxic since they break the continuity of the heritable duplex DNA and impose impediment to replication and transcription. Members of 5’nuclease excise these aberrant DNA structures during replication, repair and recombination. It is not surprising therefore that mutations in members of 5’nucleases have been linked to various disease states including cancer and aging. Furthermore, some of these nucleases are highly over-expressed in several cancers to compensate for deficiencies in their damage response pathways.

            Despite the importance of 5’nuclease it remains unclear how they recognize normal DNA sequence just based on their structure and precisely cleave them. The knowledge gap in structural studies that can access protein and DNA dynamics in 5’nucleases impairs substantially the drug development enterprises against numerous severe human cancers.

            The proposed research project within the framework of the VSPR (Visiting Student Research Internship Program) program is mainly focused on the molecular bases of substrate recognition by 5’nucleases. The project combines cutting-edge computational resources and state-of-the-art biophysical computational tools, including full-atom molecular dynamics (MD) simulations, to establish the conformational states and dynamics of bubble DNA structure and how they are influenced by the bubble size and DNA sequence. DNA bubbles structure is the key intermediary step during nucleotide excision repair that separate the strand containing the lesion site from the intact one before two members of 5’nucleases, XPG and XPF, perform two concerted cleavages to release the damage-containing ssDNA. Establishing the bubble conformer(s) will pave the way for better understand of its interaction with XPG and XPF.

            These studies will be accompanied and verified side-by-side by experimental results derived from the cutting-edge biophysical techniques, including single-molecule FRET (smFRET) and high-resolution multidimensional Nuclear Magnetic Resonance (NMR) experiments. In one alleged model, the bubble DNA structure might display dynamic conformations and the nucleases involved in NER simply capture the correct conformer. In another model the bubble might have stable conformer(s) and the nucleases actively bind to them and mold them into the ''correct'' conformer. The computational work, like full-atom molecular dynamics simulations assisted with the experiments smFRET and NMR data would help in decoding the actual mechanism of action of XPG and XPF against the bubble DNA.

            This project is suitable for students with bioscience, physics, or engineering background.

Deliverables/Expectations

We offer the students participating in this project to learn:
  • how to use the biophysical techniques and design the workload;
  • how to become self-reliant in the design, preparation, running and analysis of the molecular dynamics (MD) simulations of biologically essential macromolecular systems;
  • how to analyze and integrate the results derived from different computational techniques with the experimentally derived information from the cutting-edge biophysical techniques, including smFRET and Bio-NMR.

To complete the above tasks we expect from the students:

  • A positive approach and strong will to work in the interdisciplinary and international research team;
  • the preparation of the final report in the article format;
the preparation of presentation that will be given during the group meeting.​

Faculty Name

Mariusz Jaremko

Field of Study

​​ Structural Biology, Computational Biology, Protein-DNA interactions, FRET, NMR, Molecular Dynamics (MD), Biophysics