Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding.
PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3'-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of approximately 2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of approximately 9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed.
Yang
Ye
Y
Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Dou
Shuo-Xing
SX
Ren
Hua
H
Wang
Peng-Ye
PY
Zhang
Xing-Dong
XD
Qian
Min
M
Pan
Bing-Yi
BY
Xi
Xu Guang
XG
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C084217:pcrA protein, Bacteria; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases