RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling.
Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre-steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.
Department of Biochemistry, Momentum Motor Enzymology Research Group, Eötvös Loránd University, H-1117, Budapest, Hungary.
Kata
K
Sarlós
Máté
M
Gyimesi
Mihály
M
Kovács
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D021381:Protein Transport; D053484:RecQ Helicases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D004277:DNA, Single-Stranded; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053484:RecQ Helicases