Journal of Biological Chemistry
Volume 289, Issue 9, 28 February 2014, Pages 5938-5949
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DNA and Chromosomes
A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase*

https://doi.org/10.1074/jbc.M113.530741Get rights and content
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The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.

ATPases
DNA Repair
DNA-Protein Interaction
Molecular Motors
Protein Conformation
Recombination
Structure

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*

This work was supported by Human Frontier Science Program Grant RGY0072/2010 (to M. K.) and “Momentum” Program of the Hungarian Academy of Sciences Grant LP2011-006/2011 (to M. K.). This work was also supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of the TÁMOP 4.2.4.A/1-11-1-2012-0001 “National Excellence Program.”

1

Both authors contributed equally to this work.