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.