Negative Control of Plasmid R6K Replication: Possible Role of Intermolecular Coupling of Replication Origins

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1989 Oct 1

PMID 2682632

Citations 57

Authors

M J McEachern

M A Bott

P A Tooker

D R Helinski

Affiliations

Soon will be listed here.

Abstract

The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.

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References

Stalker D, Kolter R, Helinski D . Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979; 76(3):1150-4. PMC: 383207. DOI: 10.1073/pnas.76.3.1150. View

Kolter R, Helinski D . Construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid. 1978; 1(4):571-80. DOI: 10.1016/0147-619x(78)90014-8. View

Inuzuka N, Inuzuka M, Helinski D . Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040. J Biol Chem. 1980; 255(23):11071-4. View

Crosa J . Three origins of replication are active in vivo in the R plasmid RSF1040. J Biol Chem. 1980; 255(23):11075-7. View

LABHART P, Koller T . Electron microscope specimen preparation of rat liver chromatin by a modified Miller spreading technique. Eur J Cell Biol. 1981; 24(2):309-16. View

Stalker D, Kolter R, Helinski D . Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol. 1982; 161(1):33-43. DOI: 10.1016/0022-2836(82)90276-5. View

Shafferman A, Kolter R, Stalker D, Helinski D . Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982; 161(1):57-76. DOI: 10.1016/0022-2836(82)90278-9. View

Germino J, Bastia D . The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding. Cell. 1983; 32(1):131-40. DOI: 10.1016/0092-8674(83)90503-2. View

Shafferman A, Helinski D . Structural properties of the beta origin of replication of plasmid R6K. J Biol Chem. 1983; 258(7):4083-90. View

10.

Tsutsui H, Fujiyama A, Murotsu T, Matsubara K . Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control. J Bacteriol. 1983; 155(1):337-44. PMC: 217685. DOI: 10.1128/jb.155.1.337-344.1983. View

11.

Stalker D, Filutowicz M, Helinski D . Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication. Proc Natl Acad Sci U S A. 1983; 80(18):5500-4. PMC: 384285. DOI: 10.1073/pnas.80.18.5500. View

12.

McEachern M, Filutowicz M, Helinski D . Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 1985; 82(5):1480-4. PMC: 397286. DOI: 10.1073/pnas.82.5.1480. View

13.

Kelley W, Bastia D . Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A. 1985; 82(9):2574-8. PMC: 397606. DOI: 10.1073/pnas.82.9.2574. View

14.

Trawick J, Kline B . A two-stage molecular model for control of mini-F replication. Plasmid. 1985; 13(1):59-69. DOI: 10.1016/0147-619x(85)90056-3. View

15.

Filutowicz M, Davis G, Greener A, Helinski D . Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985; 13(1):103-14. PMC: 340977. DOI: 10.1093/nar/13.1.103. View

16.

Mukherjee S, Patel I, Bastia D . Conformational changes in a replication origin induced by an initiator protein. Cell. 1985; 43(1):189-97. DOI: 10.1016/0092-8674(85)90023-6. View

17.

Filutowicz M, Uhlenhopp E, Helinski D . Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 1986; 187(2):225-39. DOI: 10.1016/0022-2836(86)90230-5. View

18.

Filutowicz M, McEachern M, Helinski D . Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986; 83(24):9645-9. PMC: 387197. DOI: 10.1073/pnas.83.24.9645. View

19.

Mukherjee S, Erickson H, Bastia D . Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein. Cell. 1988; 52(3):375-83. DOI: 10.1016/s0092-8674(88)80030-8. View

20.

Pal S, Chattoraj D . P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol. 1988; 170(8):3554-60. PMC: 211328. DOI: 10.1128/jb.170.8.3554-3560.1988. View