The RepA protein of plasmid RSF1010 is a replicative DNA helicase.
The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (Röleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5' --> 3' polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.
Scherzinger
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E
Max-Planck-Institut für Molekulare Genetik, Dahlem, Ihnestrasse 73, D-14195 Berlin, Germany. lanka@mpimg-berlin-dahlem.mpg.de
Ziegelin
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G
Bárcena
M
M
Carazo
J M
JM
Lurz
R
R
Lanka
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E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D011756:Diphosphates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D011756:Diphosphates; D004791:Enzyme Inhibitors; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D004265:DNA Helicases