Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K (original) (raw)

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Department of Bacteriology, 1550 Linden Drive, University of Wisconsin

Madison, Wl 53706, USA

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,

Department of Bacteriology, 1550 Linden Drive, University of Wisconsin

Madison, Wl 53706, USA

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Department of Bacteriology, 1550 Linden Drive, University of Wisconsin

Madison, Wl 53706, USA

*To whom correspondence should be addressed

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Received:

04 November 1991

Revision received:

23 January 1992

Accepted:

23 January 1992

Published:

25 February 1992

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Frank Wu, llya Goldberg, Marcin Filutowicz, Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K, Nucleic Acids Research, Volume 20, Issue 4, 25 February 1992, Pages 811–817, https://doi.org/10.1093/nar/20.4.811
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Abstract

A dnaA ‘null’ strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (α,γ,β). DnaA binds In vitro to sites In two functionally distinct segments of the central γ origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt π protein are decreased or If copy-up π mutant proteins are provided In trans. DnaA does not effect expression of R6K replication initiator protein π, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes π. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the γ origin and might be sufficient and required for the activity of the α and β origins as well. Implications of the DnaA protein binding to two domains of the γ origin and the role of the 106-bp origin enhancer in replication are discussed.

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