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Positioning and the specific sequence of each 13‐mer motif are critical for activity of the plasmid RK2 replication origin
Author(s) -
Kowalczyk Lukasz,
Rajewska Magdalena,
Konieczny Igor
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04770.x
Subject(s) - biology , dnaa , plasmid , origin of replication , genetics , inverted repeat , mutant , sequence motif , ter protein , autonomously replicating sequence , gene , genome
Summary The minimal replication origin of the broad‐host‐range plasmid RK2, oriV , contains five iterons which are binding sites for the plasmid‐encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT‐rich region containing four 13‐mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13‐mer motifs have been constructed and analysed for replication activity in vivo and in vitro . The data show that the replacement of oriV 13‐mers by similar but not identical 13‐mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13‐mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single‐nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13‐mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli . Our experiments demonstrate considerable complexity in the plasmid AT‐rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence‐specific protein interactions within the 13‐mers during assembly of replication complexes at the plasmid origin.