2 research outputs found
Quantitative tool for in vivo analysis of DNA-binding proteins using High Resolution Sequencing Data
DNA-binding proteins (DBPs) such as repair proteins, DNA polymerases, re-
combinases, transcription factors, etc. manifest diverse stochastic behaviours
dependent on physiological conditions inside the cell.
Now that multiple independent in vitro studies have extensively characterised
different aspects of the biochemistry of DBPs, computational and mathematical
tools that would be able to integrate this information into a coherent framework
are in huge demand, especially when attempting a transition to in vivo characterisation of these systems.
ChIP-Seq is the method commonly used to study DBPs in vivo. This method
generates high resolution sequencing data { population scale readout of the
activity of DBPs on the DNA. The mathematical tools available for the analysis
of this type of data are at the moment very restrictive in their ability to extract
mechanistic and quantitative details on the activity of DBPs. The main trouble
that researchers experience when analysing such population scale sequencing data
is effectively disentangling complexity in these data, since the observed output
often combines diverse outcomes of multiple unsynchronised processes reflecting
biomolecular variability.
Although being a static snapshot ChIP-Seq can be effectively utilised as a readout
for the dynamics of DBPs in vivo. This thesis features a new approach to
ChIP-Seq analysis { namely accessing the concealed details of the dynamic
behaviour of DBPs on DNA using probabilistic modelling, statistical inference
and numerical optimisation. In order to achieve this I propose to integrate
previously acquired assumptions about the behaviour of DBPs into a Markov-
Chain model which would allow to take into account their intrinsic stochasticity.
By incorporating this model into a statistical model of data acquisition, the
experimentally observed output can be simulated and then compared to in vivo
data to reverse engineer the stochastic activity of DBPs on the DNA.
Conventional tools normally employ simple empirical models where the parameters have no link with the mechanistic reality of the process under scrutiny. This
thesis marks the transition from qualitative analysis to mechanistic modelling in
an attempt to make the most of the high resolution sequencing data.
It is also worth noting that from a computer science point of view DBPs are
of great interest since they are able to perform stochastic computation on DNA
by responding in a probabilistic manner to the patterns encoded in the DNA.
The theoretical framework proposed here allows to quantitatively characterise
complex responses of these molecular machines to the sequence features
RecG directs DNA synthesis during double-strand break repair
Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability