THE REPA PROTEIN OF PLASMID RSF1010 IS A REPLICATIVE DNA HELICASE

The RepA protein of the mobilizable broad host range plasmid RSF1010 h as a key function in its replication. RepA is one of the smallest know n helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of Re pA. Gel filtration and cross-linking experiments demonstrated the hexa meric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from Rep A crystals diffracting at 3.5-Angstrom resolution (Roleke, 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 prefer s 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 fue led by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, a nd pyrophosphate inhibit the helicase activity, whereas inorganic phos phate does not. The presence of Escherichia coli single-stranded DNA-b inding protein stimulates unwinding at physiological pH 2-3-fold, wher eas the RSF1010 replicon-specific primase, RepB' protein, has no effec t, either in the presence or in the absence of single-stranded DNA-bin ding protein.