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PMID: 14765124 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S.

DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding.

The EMBO journal ·Vol. 23 ·No. 4 ·2004-02-25 ·Pages 897-907

Remus D, Beall EL, Botchan MR

Abstract

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.

MeSH Terms
Animals Base Sequence Binding Sites DNA, Superhelical/chemistry DNA-Binding Proteins/chemistry Drosophila Proteins/chemistry Drosophila melanogaster Electrophoretic Mobility Shift Assay Nucleic Acid Conformation Origin Recognition Complex Plasmids Replication Origin
Chemicals
DNA, Superhelical DNA-Binding Proteins Drosophila Proteins Origin Recognition Complex
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Remus Dirk
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720-3204, USA.
Beall Eileen L
Botchan Michael R
References (43)
43 references, click to expand
  1. Eukaryotic DNA replication origins: reconciling disparate data.
    Cell. 2003 Aug 8;114(3):274-5 PMID: 12914691
  2. Sequence-independent DNA binding and replication initiation by the human origin recognition complex.
    Genes Dev. 2003 Aug 1;17(15):1894-908 PMID: 12897055
  3. Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis.
    Proc Natl Acad Sci U S A. 1975 Dec;72(12):4876-80 PMID: 174079
  4. Regulated replication of DNA microinjected into eggs of Xenopus laevis.
    Cell. 1980 Oct;21(3):761-71 PMID: 6254667
  5. Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.
    Proc Natl Acad Sci U S A. 1983 Oct;80(19):5817-21 PMID: 6310593
  6. Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast.
    Cell. 1984 Aug;38(1):55-64 PMID: 6380762
  7. On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli.
    J Biol Chem. 1986 Jan 25;261(3):1025-30 PMID: 3511041
  8. Both DNA topoisomerases I and II relax 2 micron plasmid DNA in living yeast cells.
    Cell. 1986 Apr 11;45(1):65-70 PMID: 3006927
  9. Initiation protein induced helix destabilization at the lambda origin: a prepriming step in DNA replication.
    Cell. 1988 Feb 12;52(3):385-95 PMID: 2830983
  10. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome.
    Cell. 1988 Mar 11;52(5):743-55 PMID: 2830993
  11. Regulation of DNA replication during Drosophila development.
    Annu Rev Genet. 1987;21:373-403 PMID: 3327470
  12. Effect of transcription of yeast chromatin on DNA topology in vivo.
    EMBO J. 1990 Jun;9(6):1873-81 PMID: 1693332
  13. DNA binding properties of the purified Antennapedia homeodomain.
    Proc Natl Acad Sci U S A. 1990 Jun;87(11):4093-7 PMID: 1971945
  14. A yeast chromosomal origin of DNA replication defined by multiple functional elements.
    Science. 1992 Feb 14;255(5046):817-23 PMID: 1536007
  15. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.
    Nature. 1992 May 14;357(6374):128-34 PMID: 1579162
  16. Protein-DNA interactions at a yeast replication origin.
    Nature. 1992 May 14;357(6374):169-72 PMID: 1579168
  17. Transition in specification of embryonic metazoan DNA replication origins.
    Science. 1995 Nov 10;270(5238):994-7 PMID: 7481806
  18. Autonomous replication in Drosophila melanogaster tissue culture cells.
    Chromosoma. 1995 May;103(9):597-605 PMID: 7587582
  19. Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.
    EMBO J. 1995 Dec 15;14(24):6348-57 PMID: 8557055
  20. Regulation of replicon size in Xenopus egg extracts.
    Science. 1997 Feb 14;275(5302):993-5 PMID: 9020085
  21. Large scale preparation of positively supercoiled DNA using the archaeal histone HMf.
    Nucleic Acids Res. 1997 Apr 15;25(8):1660-1 PMID: 9092677
  22. Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApolalpha-dE2F locus of Drosophila melanogaster.
    Mol Cell Biol. 1999 Jan;19(1):547-55 PMID: 9858578
  23. Replication origins in metazoan chromosomes: fact or fiction?
    Bioessays. 1999 Jan;21(1):5-16 PMID: 10070250
  24. The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.
    Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2656-61 PMID: 10077566
  25. Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.
    Genes Dev. 1999 May 15;13(10):1289-96 PMID: 10346817
  26. Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.
    Methods. 1999 Jul;18(3):407-17 PMID: 10455001
  27. Identification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe.
    Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12367-72 PMID: 10535928
  28. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element.
    Genes Dev. 1999 Oct 15;13(20):2639-49 PMID: 10541550
  29. Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro.
    Genes Dev. 2000 Jul 1;14(13):1631-41 PMID: 10887157
  30. Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.
    EMBO J. 2000 Sep 1;19(17):4774-82 PMID: 10970868
  31. Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times.
    J Cell Biol. 2001 Jan 8;152(1):15-25 PMID: 11149917
  32. Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle.
    EMBO J. 2001 Aug 1;20(15):4263-77 PMID: 11483529
  33. Making sense of eukaryotic DNA replication origins.
    Science. 2001 Oct 5;294(5540):96-100 PMID: 11588251
  34. Functional analysis of mutant and wild-type Drosophila origin recognition complex.
    Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):11997-2002 PMID: 11593009
  35. Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit.
    Mol Cell Biol. 2001 Dec;21(23):8095-103 PMID: 11689699
  36. The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein.
    Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13589-94 PMID: 11717425
  37. The origin recognition complex: from simple origins to complex functions.
    Genes Dev. 2002 Mar 15;16(6):659-72 PMID: 11914271
  38. The origin recognition complex marks a replication origin in the human TOP1 gene promoter.
    J Biol Chem. 2002 Aug 30;277(35):31430-40 PMID: 12004060
  39. The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation.
    EMBO J. 2002 Sep 16;21(18):4763-73 PMID: 12234917
  40. Motors and switches: AAA+ machines within the replisome.
    Nat Rev Mol Cell Biol. 2002 Nov;3(11):826-35 PMID: 12415300
  41. Role for a Drosophila Myb-containing protein complex in site-specific DNA replication.
    Nature. 2002 Dec 19-26;420(6917):833-7 PMID: 12490953
  42. Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem.
    Bioessays. 2003 Feb;25(2):116-25 PMID: 12539237
  43. Lac repressor binding to synthetic DNAs of defined nucleotide sequence.
    Proc Natl Acad Sci U S A. 1972 Mar;69(3):761-4 PMID: 4501591
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1038/sj.emboj.7600077
Published
2004-02-25
Epub
2004-00-05
Pages
897-907
Language
English
Region
England
NLM ID
8208664
PMCID
PMC380993
Subset
IM
Grants
NCI NIH HHS · R01 CA030490 · United States
NCI NIH HHS · R37 CA030490 · United States
NCI NIH HHS · CA-30490 · United States
External Links
PubMed source DOI: 10.1038/sj.emboj.7600077 PMC Full Text View journal details
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