DNA helicases play an essential role in facilitating cellular DNA metabolisms
including genome replication, DNA repair, recombination, transcription as well as
telomere maintenance. The RecQ family of DNA helicases has received much
attention in the past few years. It is very well proven that RecQ family helicases play
important roles in coordinating genome maintenance pathways in living cells. In the
absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including
increased mitotic recombination, elevated chromosome missegregation,
hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three
different RecQ helicase family members give rise to human disorders leading to
genome instability and cancer susceptibility: Bloom syndrome (BS), Werner
syndrome (WRN), and Rothmund-Thomson syndrome.
For 10 years, our group has been studying the molecular mechanisms of RecQ
helicases in combination of different methods from biochemistry to physics. We have
shown that while E. coli RecQ helicase function as a monomer, the superfamily 1
helicase Bacillus stearothermophilus PcrA is a dimmer in solution. We also
demonstrated that an evolutionary conserved zinc finger in RecQ family helicase
plays essential roles in DNA binding, protein folding and DNA substrate recognition
and discrimination. These results shed new light on the mechanism transmitting
chemical energy from ATP hydrolysis to DNA unwinding. By analyzing diseasecausing
missence mutations in Bloom syndrome protein, we have provided insights
into the molecular elements implicated in coupling DNA binding to ATP hydrolysis
and DNA unwinding. Our finding will help to explain the mechanism underlying
BLM catalysis and interpreting new BLM causing mutations identified in the future.
Our previous experiment performed with single-molecule assay has revealed
strand switching phenomenon. Recently, we have shown that the unwinding rate of E.
coli UvrD decreases exponentially with increasing DNA-destabilizing force. This
result conflicts with expectation and observations in recent papers on the topic.
However, this surprising result could give a deep insight into the molecular
mechanism of UvrD mediated-DNA unwinding. In fact, while crystal structure studies
have revealed that some helicases such as PcrA and UvrD unwinding DNA with one
bp step size, all of biochemical bulk assays show that the step size of these enzymes
varies between 4 and 6 bp. Based on our experimental data and in combination with
the data obtained from the crystal structure and kinetic studies, we proposed a new
model to explain the mysterious conflict.

Invité par Philippe Fossé (59 99)

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