A Nucleotide- and HRDC-domain-dependent Structural Transition in DNA-bound RecQ Helicase [Enzymology]

January 8th, 2014 by Kocsis, Z. S., Sarlos, K., Harami, G. M., Martina, M., Kovacs, M.

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded (ss) DNA and, in turn, the restructuring of multi-stranded DNA substrates during genome maintenance processes. In this study we used the tryptophan fluorescence signal of E. 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 HRDC domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multi-stranded DNA processing by RecQ helicases.