Open AccessDetermination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle‐replicating plasmids
Author(s) -
Grohmann Elisabeth,
Zechner Ellen L,
Espinosa Manuel
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb10453.x
Subject(s) - plasmid , rolling circle replication , biology , bacillus subtilis , dna , escherichia coli , dna replication , microbiology and biotechnology , origin of replication , nucleotide , genetics , gene , bacteria
Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so‐called nic site is catalyzed by plasmid‐encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae , respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double‐stranded origin of the rolling circle‐replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.