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Mol Cell Biol. 1992 September; 12(9): 4056–4066.
PMCID: PMC360298

Replication forks pause at yeast centromeres.

Abstract

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.

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Selected References

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  • Baker RE, Fitzgerald-Hayes M, O'Brien TC. Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site. J Biol Chem. 1989 Jun 25;264(18):10843–10850. [PubMed]
  • Bedinger P, Hochstrasser M, Jongeneel CV, Alberts BM. Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule. Cell. 1983 Aug;34(1):115–123. [PubMed]
  • Bell GI, DeGennaro LJ, Gelfand DH, Bishop RJ, Valenzuela P, Rutter WJ. Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs. J Biol Chem. 1977 Nov 25;252(22):8118–8125. [PubMed]
  • Bloom KS, Carbon J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell. 1982 Jun;29(2):305–317. [PubMed]
  • Bonne-Andrea C, Wong ML, Alberts BM. In vitro replication through nucleosomes without histone displacement. Nature. 1990 Feb 22;343(6260):719–726. [PubMed]
  • Bram RJ, Kornberg RD. Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor. Mol Cell Biol. 1987 Jan;7(1):403–409. [PMC free article] [PubMed]
  • Brewer BJ. When polymerases collide: replication and the transcriptional organization of the E. coli chromosome. Cell. 1988 Jun 3;53(5):679–686. [PubMed]
  • Brewer BJ, Fangman WL. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. [PubMed]
  • Brewer BJ, Fangman WL. A replication fork barrier at the 3' end of yeast ribosomal RNA genes. Cell. 1988 Nov 18;55(4):637–643. [PubMed]
  • Cai MJ, Davis RW. Purification of a yeast centromere-binding protein that is able to distinguish single base-pair mutations in its recognition site. Mol Cell Biol. 1989 Jun;9(6):2544–2550. [PMC free article] [PubMed]
  • Carbon J, Clarke L. Structural and functional analysis of a yeast centromere (CEN3). J Cell Sci Suppl. 1984;1:43–58. [PubMed]
  • Carbon J, Clarke L. Centromere structure and function in budding and fission yeasts. New Biol. 1990 Jan;2(1):10–19. [PubMed]
  • Clarke L, Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. [PubMed]
  • Cumberledge S, Carbon J. Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae. Genetics. 1987 Oct;117(2):203–212. [PubMed]
  • de Massy B, Béjar S, Louarn J, Louarn JM, Bouché JP. Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1759–1763. [PubMed]
  • Densmore L, Payne WE, Fitzgerald-Hayes M. In vivo genomic footprint of a yeast centromere. Mol Cell Biol. 1991 Jan;11(1):154–165. [PMC free article] [PubMed]
  • Dhar V, Schildkraut CL. Role of EBNA-1 in arresting replication forks at the Epstein-Barr virus oriP family of tandem repeats. Mol Cell Biol. 1991 Dec;11(12):6268–6278. [PMC free article] [PubMed]
  • Fangman WL, Brewer BJ. Activation of replication origins within yeast chromosomes. Annu Rev Cell Biol. 1991;7:375–402. [PubMed]
  • Fitzgerald-Hayes M, Clarke L, Carbon J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell. 1982 May;29(1):235–244. [PubMed]
  • Gahn TA, Schildkraut CL. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell. 1989 Aug 11;58(3):527–535. [PubMed]
  • Gaudet A, Fitzgerald-Hayes M. Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):68–75. [PMC free article] [PubMed]
  • Hegemann JH, Pridmore RD, Schneider R, Philippsen P. Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres. Mol Gen Genet. 1986 Nov;205(2):305–311. [PubMed]
  • Hegemann JH, Shero JH, Cottarel G, Philippsen P, Hieter P. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2523–2535. [PMC free article] [PubMed]
  • Hidaka M, Akiyama M, Horiuchi T. A consensus sequence of three DNA replication terminus sites on the E. coli chromosome is highly homologous to the terR sites of the R6K plasmid. Cell. 1988 Nov 4;55(3):467–475. [PubMed]
  • Hill TM, Henson JM, Kuempel PL. The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1754–1758. [PubMed]
  • Hill TM, Kopp BJ, Kuempel PL. Termination of DNA replication in Escherichia coli requires a trans-acting factor. J Bacteriol. 1988 Feb;170(2):662–668. [PMC free article] [PubMed]
  • Hill TM, Pelletier AJ, Tecklenburg ML, Kuempel PL. Identification of the DNA sequence from the E. coli terminus region that halts replication forks. Cell. 1988 Nov 4;55(3):459–466. [PubMed]
  • Hsiao CL, Carbon J. Direct selection procedure for the isolation of functional centromeric DNA. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3760–3764. [PubMed]
  • Huberman JA, Spotila LD, Nawotka KA, el-Assouli SM, Davis LR. The in vivo replication origin of the yeast 2 microns plasmid. Cell. 1987 Nov 6;51(3):473–481. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Jiang WD, Philippsen P. Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA. Mol Cell Biol. 1989 Dec;9(12):5585–5593. [PMC free article] [PubMed]
  • Johnston LH, Williamson DH. An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1978 Aug 17;164(2):217–225. [PubMed]
  • Jongeneel CV, Formosa T, Munn M, Alberts BM. Enzymological studies of the T4 replication proteins. Adv Exp Med Biol. 1984;179:17–33. [PubMed]
  • Krysan PJ, Calos MP. Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol Cell Biol. 1991 Mar;11(3):1464–1472. [PMC free article] [PubMed]
  • Lechner J, Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. [PubMed]
  • Linskens MH, Huberman JA. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4927–4935. [PMC free article] [PubMed]
  • Mann C, Davis RW. Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jan;6(1):241–245. [PMC free article] [PubMed]
  • Levitt M. Real-time interactive frequency filtering of molecular dynamics trajectories. J Mol Biol. 1991 Jul 5;220(1):1–4. [PubMed]
  • McCarroll RM, Fangman WL. Time of replication of yeast centromeres and telomeres. Cell. 1988 Aug 12;54(4):505–513. [PubMed]
  • McGrew J, Diehl B, Fitzgerald-Hayes M. Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):530–538. [PMC free article] [PubMed]
  • Mellor J, Jiang W, Funk M, Rathjen J, Barnes CA, Hinz T, Hegemann JH, Philippsen P. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 1990 Dec;9(12):4017–4026. [PubMed]
  • Mirkovitch J, Mirault ME, Laemmli UK. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. [PubMed]
  • Newlon CS. Yeast chromosome replication and segregation. Microbiol Rev. 1988 Dec;52(4):568–601. [PMC free article] [PubMed]
  • Newlon CS, Lipchitz LR, Collins I, Deshpande A, Devenish RJ, Green RP, Klein HL, Palzkill TG, Ren RB, Synn S, et al. Analysis of a circular derivative of Saccharomyces cerevisiae chromosome III: a physical map and identification and location of ARS elements. Genetics. 1991 Oct;129(2):343–357. [PubMed]
  • Ng R, Carbon J. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Dec;7(12):4522–4534. [PMC free article] [PubMed]
  • Palmer DK, O'Day K, Trong HL, Charbonneau H, Margolis RL. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3734–3738. [PubMed]
  • Palzkill TG, Newlon CS. A yeast replication origin consists of multiple copies of a small conserved sequence. Cell. 1988 May 6;53(3):441–450. [PubMed]
  • Panzeri L, Landonio L, Stotz A, Philippsen P. Role of conserved sequence elements in yeast centromere DNA. EMBO J. 1985 Jul;4(7):1867–1874. [PubMed]
  • Rawlins DR, Milman G, Hayward SD, Hayward GS. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell. 1985 Oct;42(3):859–868. [PubMed]
  • Reynolds AE, McCarroll RM, Newlon CS, Fangman WL. Time of replication of ARS elements along yeast chromosome III. Mol Cell Biol. 1989 Oct;9(10):4488–4494. [PMC free article] [PubMed]
  • Saffer LD, Miller OL., Jr Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication. Mol Cell Biol. 1986 Apr;6(4):1148–1157. [PMC free article] [PubMed]
  • Saunders M, Fitzgerald-Hayes M, Bloom K. Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci U S A. 1988 Jan;85(1):175–179. [PubMed]
  • Saunders MJ, Yeh E, Grunstein M, Bloom K. Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres. Mol Cell Biol. 1990 Nov;10(11):5721–5727. [PMC free article] [PubMed]
  • Smith GE, Summers MD. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. [PubMed]
  • Struhl K, Stinchcomb DT, Scherer S, Davis RW. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. [PubMed]
  • Surosky RT, Tye BK. Resolution of dicentric chromosomes by Ty-mediated recombination in yeast. Genetics. 1985 Jul;110(3):397–419. [PubMed]
  • Van Houten JV, Newlon CS. Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III. Mol Cell Biol. 1990 Aug;10(8):3917–3925. [PMC free article] [PubMed]
  • Widom J. Toward a unified model of chromatin folding. Annu Rev Biophys Biophys Chem. 1989;18:365–395. [PubMed]

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