Nucleic Acids Research, 1992, Vol. 20, No. 1 41-48
© 1992
ENZYMOLOGY |
In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins
Max-Planck-Institut für Molekulare Genetik Abteilung Schuster, Ihnestrasse 73, D-1000 Berlin 33, FRG
Received October 29, 1991. Accepted December 5, 1991.
We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. On the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DMA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SOS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.
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