The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT.
The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5'-end and the 3'-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5'-GpT-3' dinucleotide, within the sequence 5'-TAGTGTG/TTA-3'. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.
Guzmán
L M
LM
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas Velázquez, Madrid, Spain.
Espinosa
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria; D004264:DNA Topoisomerases, Type I