We investigate the use of physical modeling to extract
mechanistic details from quantitative biological data,
with a focus on the physical properties of nucleic acids.
It is well understood that DNA stores
genetic information, RNA acts as a carrier of
this information, and that both must interact with
a wide array of protein complexes in order to perform these functions.
However, the physical mechanisms by which
these interactions occur are much less clear.
For example, Protein-bound duplex DNA is often bent or kinked. Yet, quantification of
intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA
cyclization experiments have indicated that DNA may form sharp bends more easily than
predicted by the established worm-like chain (WLC) model.
One proposed explanation suggests that local melting of a
few base pairs introduces flexible hinges.
We test this model for three sequences at temperatures
from 23C to 65C. We find that small
melted bubbles are significantly more flexible
than double-stranded DNA and can alter
DNA flexibility at physiological temperatures.
There are also many important proteins which bind single-stranded nucleic acids, such
as the nucleocapsid protein in HIV and the RecA DNA repair protein in bacteria.
The presence of such proteins can strongly alter the secondary structure of the
nucleic acid molecules. Therefore, accurate modeling of the interaction
between single-stranded nucleic acids and such proteins
is essential to fully understanding many biological processes.
We develop a model for predicting nucleic acid secondary structure in the presence
of single stranded binding proteins, and implement it as an extension of the
Vienna RNA Package. Using this model we are
able to predict the probability of the protein binding at any position
in the nucleic acid sequence, the impact of the protein on nucleic acid
base pairing, the end-to-end distance distribution for the nucleic
acid, and FRET distributions for fluorophores attached to the nucleic acid.
Eukaryotic DNA also interacts strongly with nucleosome protein complexes,
which wrap and compact this DNA.
The expression, replication and repair of DNA requires
nucleosomes to be unwrapped and disassembled.
We have developed a quantitative model of nucleosome
dynamics and calibrated this model using results from high precision
single molecule nucleosome unzipping experiments.
We then tested its predictions for
experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex
hMSH2-hMSH6. We found that this calibrated model
quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate
DNA sequences and four distinct histone modification states. In addition, this model provides
mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone
modifications on this disassembly reaction.
We also found that this model accurately predicts the rate at
which lexA is able to trap nucleosome unwrapping fluctuations.
This model's precise agreement with current
experiments suggests that it can be applied more generally to
provide important mechanistic understanding of the numerous
nucleosome alterations that occur during DNA processing.