Replication origin of the broad host range plasmid RK2. Positioning of various motifs is critical for initiation of replication.

The Journal of Biological Chemistry
K S DoranD R Helinski

Abstract

The 393-base pair minimal origin, oriV, of plasmid RK2 contains three iterated motifs essential for initiation of replication: consensus sequences for binding the bacterial DnaA protein, DnaA boxes, which have recently been shown to bind the DnaA protein; 17-base pair direct repeats, iterons, which bind the plasmid encoded replication protein, TrfA; and A + T-rich repeated sequences, 13-mers, which serve as the initial site of helix destabilization. To investigate how the organization of the RK2 origin contributes to the mechanism of replication initiation, mutations were introduced into the minimal origin which altered the sequence and/or spacing of each particular region relative to the rest of the origin. These altered origins were analyzed for replication activity in vivo and in vitro, for localized strand opening and for DnaB helicase mediated unwinding. Mutations in the region between the iterons and the 13-mers which altered the helical phase or the intrinsic DNA curvature prevented strand opening of the origin and consequently abolished replication activity. Insertions of more or less than one helical turn between the DnaA boxes and the iterons also inactivated the replication origin. In these mutants, however, strand o...Continue Reading

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Citations

Jan 14, 2003·EMBO Reports·Igor Konieczny
Jul 27, 1999·Molecular Microbiology·K S DoranI Konieczny
Oct 9, 2002·Molecular Microbiology·Beatriz MaestroElena Fernández-Tresguerres
Aug 8, 2008·Proceedings of the National Academy of Sciences of the United States of America·Magdalena RajewskaIgor Konieczny
Feb 4, 2003·Journal of Bacteriology·Beatriz MaestroElena Fernández-Tresguerres
Jan 6, 1999·Proceedings of the National Academy of Sciences of the United States of America·H J DattaD Bastia
Jul 2, 2003·Proceedings of the National Academy of Sciences of the United States of America·Yong JiangAresa Toukdarian
Dec 3, 2014·Plasmid·Elzbieta ZabrockaIgor Konieczny
Jul 22, 2015·Proceedings of the National Academy of Sciences of the United States of America·Aleksandra WawrzyckaIgor Konieczny
Jul 6, 2010·Plasmid·Katarzyna KolatkaIgor Konieczny
Nov 19, 2011·FEMS Microbiology Reviews·Magdalena RajewskaIgor Konieczny
Aug 27, 2016·Frontiers in Molecular Biosciences·Katarzyna E WegrzynIgor Konieczny
Oct 18, 2016·Frontiers in Molecular Biosciences·Jyoti K JhaDhruba K Chattoraj
Mar 13, 2002·The Journal of Biological Chemistry·Igor Konieczny, Krzysztof Liberek
Aug 10, 2000·Molecular Microbiology·D K Chattoraj
Jun 25, 2015·Microbiology Spectrum·Igor KoniecznyKatarzyna Wegrzyn

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