A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase

Zsuzsa S Kocsis,Kata Sarlós,Gábor M Harami,Máté Martina,Mihály Kovács

doi:10.1074/jbc.m113.530741

Zsuzsa S Kocsis, Kata Sarlós + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m113.530741

Copy DOI

Abstract

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.

Highlights

The mechanistic role of DNA-induced structural changes in RecQ helicases is largely unexplored
DNA Binding by RecQ Leads to a Structural Transition Affected by the Nucleotide State of the Helicase—To mimic the nucleotide states adopted by RecQ helicase during the ATPase cycle, we used a series of nucleotides and nucleotide analogs, including ATP, ADP, AMPPNP, and ADP-AlF4
Overall DNA Binding Properties Deduced from Trp Fluorescence Data—We found the Trp fluorescence emission of RecQ helicase to be sensitive to the interaction of the enzyme with single-stranded DNA (ssDNA) and mononucleotide substrates and analogs (Fig. 1A and Tables 1–3)

Summary

Background

The mechanistic role of DNA-induced structural changes in RecQ helicases is largely unexplored. We used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. 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. Alignment of the crystal structure of human RECQ1 with those of DNA-bound complexes of other helicases provided no indications for significant DNA-induced conformational changes [32, 33]. Kinetic data on the DNA interaction or the coupling of nucleotide and DNA interactions of RecQ helicases are missing, except for the implication that in human BLM, ATP binding was largely unaffected by the presence of ssDNA [15]. We used Trp fluorescence signals in spectroscopic and rapid kinetic experiments to determine the kinetic mechanism of the interaction of the enzyme with ssDNA substrates

EXPERIMENTAL PROCEDURES

RESULTS

52 Ϯ 2 19 Ϯ 2 13 Ϯ 2

DISCUSSION