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Nucleic Acids Research, 1987, Vol. 15, No. 10 4085-4097
© 1987

Articles

Cleavage of single-stranded DNA by plasmid pT 181-encoded RepC protein

Richard R. Koepsel and Saleem A. Khan*

Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh, School of Medicine Pittsburgh, PA 15261, USA

* To whom correspondence should be addressed.

Received October 4, 1986. Revised April 14, 1987. Accepted April 14, 1987.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism, RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.


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