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. 1996 May 15;24(10):1936–1942. doi: 10.1093/nar/24.10.1936

Initiator protein pi can bind independently to two domains of the gamma origin core of plasmid R6K: the direct repeats and the A+T-rich segment.

I Levchenko 1, M Filutowicz 1
PMCID: PMC145866  PMID: 8657577

Abstract

The pi protein of plasmid R6K functions in both replication and transcription. pi autoregulates its own synthesis and is required for replication of the RISK gamma origin. pi performs these functions by binding to specific DNA sites arranged as pairs of 6-10 bp inverted repeats (IRs) or as a cluster of seven tandem 22 bp direct repeats (DRs) which lack symmetry. The sites share the TGAGRG nucleotide motif (where R is A or G). The DRs and IRs flank the central A+T-rich segment of the gamma origin. In this work we carried out DNase I and hydroxyl radical protection experiments on various deletion derivatives of the gamma origin complexed with pi protein. These experiments revealed binding of pi to a novel site embedded within the A+T-rich segment. This interaction manifests primarily by the appearance of the enhanced scissions of DNA by DNase I and hydroxyl radicals. pi interaction with the A+T-rich site is independent of pi binding to the DRs and IRs. We propose that pi protein can recognize distinct families of DNA sequences in the gamma origin.

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Selected References

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  1. Bramhill D., Kornberg A. A model for initiation at origins of DNA replication. Cell. 1988 Sep 23;54(7):915–918. doi: 10.1016/0092-8674(88)90102-x. [DOI] [PubMed] [Google Scholar]
  2. Dellis S., Feng J., Filutowicz M. Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site. J Mol Biol. 1996 Apr 5;257(3):550–560. doi: 10.1006/jmbi.1996.0184. [DOI] [PubMed] [Google Scholar]
  3. Dellis S., Filutowicz M. Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein. J Bacteriol. 1991 Feb;173(3):1279–1286. doi: 10.1128/jb.173.3.1279-1286.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Filutowicz M., Appelt K. The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. Nucleic Acids Res. 1988 May 11;16(9):3829–3843. doi: 10.1093/nar/16.9.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Filutowicz M., Davis G., Greener A., Helinski D. R. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. doi: 10.1093/nar/13.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Filutowicz M., Inman R. A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K. J Biol Chem. 1991 Dec 15;266(35):24077–24083. [PubMed] [Google Scholar]
  7. Filutowicz M., McEachern M. J., Helinski D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9645–9649. doi: 10.1073/pnas.83.24.9645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Filutowicz M., Uhlenhopp E., Helinski D. R. Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 1986 Jan 20;187(2):225–239. doi: 10.1016/0022-2836(86)90230-5. [DOI] [PubMed] [Google Scholar]
  9. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  10. Germino J., Bastia D. The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding. Cell. 1983 Jan;32(1):131–140. doi: 10.1016/0092-8674(83)90503-2. [DOI] [PubMed] [Google Scholar]
  11. Greener A., Filutowicz M. S., McEachern M. J., Helinski D. R. N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. Mol Gen Genet. 1990 Oct;224(1):24–32. doi: 10.1007/BF00259447. [DOI] [PubMed] [Google Scholar]
  12. Inuzuka M., Helinski D. R. Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5381–5385. doi: 10.1073/pnas.75.11.5381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kelley W. L., Bastia D. Conformational changes induced by integration host factor at origin gamma of R6K and copy number control. J Biol Chem. 1991 Aug 25;266(24):15924–15937. [PubMed] [Google Scholar]
  14. Kelley W. L., Patel I., Bastia D. Structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid R6K. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5078–5082. doi: 10.1073/pnas.89.11.5078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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 May;82(9):2574–2578. doi: 10.1073/pnas.82.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim Y. J., Meyer R. J. An essential iteron-binding protein required for plasmid R1162 replication induces localized melting within the origin at a specific site in AT-rich DNA. J Bacteriol. 1991 Sep;173(17):5539–5545. doi: 10.1128/jb.173.17.5539-5545.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kolter R., Helinski D. R. Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin. J Mol Biol. 1982 Oct 15;161(1):45–56. doi: 10.1016/0022-2836(82)90277-7. [DOI] [PubMed] [Google Scholar]
  18. Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
  19. Leung P. C., Teplow D. B., Harshey R. M. Interaction of distinct domains in Mu transposase with Mu DNA ends and an internal transpositional enhancer. Nature. 1989 Apr 20;338(6217):656–658. doi: 10.1038/338656a0. [DOI] [PubMed] [Google Scholar]
  20. Marians K. J. Prokaryotic DNA replication. Annu Rev Biochem. 1992;61:673–719. doi: 10.1146/annurev.bi.61.070192.003325. [DOI] [PubMed] [Google Scholar]
  21. McEachern M. J., Bott M. A., Tooker P. A., Helinski D. R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942–7946. doi: 10.1073/pnas.86.20.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McEachern M. J., Filutowicz M., Helinski D. R. 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 Mar;82(5):1480–1484. doi: 10.1073/pnas.82.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moitoso de Vargas L., Pargellis C. A., Hasan N. M., Bushman E. W., Landy A. Autonomous DNA binding domains of lambda integrase recognize two different sequence families. Cell. 1988 Sep 23;54(7):923–929. doi: 10.1016/0092-8674(88)90107-9. [DOI] [PubMed] [Google Scholar]
  24. Shafferman A., Kolter R., Stalker D., Helinski D. R. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. doi: 10.1016/0022-2836(82)90278-9. [DOI] [PubMed] [Google Scholar]
  25. Sigman D. S., Chen C. H. Chemical nucleases: new reagents in molecular biology. Annu Rev Biochem. 1990;59:207–236. doi: 10.1146/annurev.bi.59.070190.001231. [DOI] [PubMed] [Google Scholar]
  26. Stalker D. M., Kolter R., Helinski D. R. Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1150–1154. doi: 10.1073/pnas.76.3.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tullius T. D., Dombroski B. A., Churchill M. E., Kam L. Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 1987;155:537–558. doi: 10.1016/0076-6879(87)55035-2. [DOI] [PubMed] [Google Scholar]
  28. Urh M., Flashner Y., Shafferman A., Filutowicz M. Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K. J Bacteriol. 1995 Dec;177(23):6732–6739. doi: 10.1128/jb.177.23.6732-6739.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu F., Levchenko I., Filutowicz M. A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J Bacteriol. 1995 Nov;177(22):6338–6345. doi: 10.1128/jb.177.22.6338-6345.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. York D., Filutowicz M. Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. J Biol Chem. 1993 Oct 15;268(29):21854–21861. [PubMed] [Google Scholar]
  31. Yung B. Y., Kornberg A. The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli. J Biol Chem. 1989 Apr 15;264(11):6146–6150. [PubMed] [Google Scholar]

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