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Nucleic Acids Res. 1997 December 15; 25(24): 5057–5064.
PMCID: PMC147147

Functional analysis of a replication origin from Saccharomyces cerevisiae: identification of a new replication enhancer.


Yeast replication origins have a modular arrangement of essential DNA sequences containing the ARS consensus sequence (ACS) flanked by auxiliary DNA elements which stimulate origin function. One of the auxiliary elements identified at several origins is a DNA replication enhancer that binds the Abf1p protein. We have isolated an ARS sequence from Saccharomyces cerevisiae based on its ability to bind Abf1p. Here we present a detailed molecular dissection of this ARS, designated ARS 1501, and we demonstrate that it functions as a genomic replication origin on chromosome XV . Mutagenesis of the Abf1p DNA-binding sites revealed that these sequences did not contribute significantly to ARS function. Instead, a new DNA element important for replication, designated REN1501, has been located 5' to the T-rich strand of the ACS. We show that REN1501 functions in either orientation and at variable distances from the ACS, defining this element as a DNA replication enhancer. Most significantly, point mutations within this element decreased the stability of plasmids bearing ARS 1501, suggesting that REN1501 binds a protein important for replication initiation. Only three elements found at origins are known to specifically bind proteins. These include the ARS essential sequences and the Abf1p and Rap1p DNA-binding sites. We show that the function of REN1501 at the origin cannot be replaced by a Rap1p DNA-binding site or a site that binds the transcriptional factor Gal4p and can only be partially substituted for by an Abf1p recognition sequence. This implies that the role of the REN1501 element at the ARS 1501 origin is specific, and suggest that the frequency of origin firing in eukaryotic cells may be regulated by origin-specific enhancers.

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Selected References

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  • Stinchcomb DT, Struhl K, Davis RW. Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979 Nov 1;282(5734):39–43. [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]
  • 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]
  • Huberman JA, Zhu JG, Davis LR, Newlon CS. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Jul 25;16(14A):6373–6384. [PMC free article] [PubMed]
  • Fangman WL, Brewer BJ. Activation of replication origins within yeast chromosomes. Annu Rev Cell Biol. 1991;7:375–402. [PubMed]
  • Walker SS, Malik AK, Eisenberg S. Analysis of the interactions of functional domains of a nuclear origin of replication from Saccharomyces cerevisiae. Nucleic Acids Res. 1991 Nov 25;19(22):6255–6262. [PMC free article] [PubMed]
  • Rivier DH, Rine J. An origin of DNA replication and a transcription silencer require a common element. Science. 1992 May 1;256(5057):659–663. [PubMed]
  • Deshpande AM, Newlon CS. The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4305–4313. [PMC free article] [PubMed]
  • Marahrens Y, Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. [PubMed]
  • Theis JF, Newlon CS. Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function. Mol Cell Biol. 1994 Nov;14(11):7652–7659. [PMC free article] [PubMed]
  • Huang RY, Kowalski D. Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome. Nucleic Acids Res. 1996 Mar 1;24(5):816–823. [PMC free article] [PubMed]
  • Shore D, Stillman DJ, Brand AH, Nasmyth KA. Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. EMBO J. 1987 Feb;6(2):461–467. [PubMed]
  • Eisenberg S, Civalier C, Tye BK. Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences. Proc Natl Acad Sci U S A. 1988 Feb;85(3):743–746. [PubMed]
  • Sweder KS, Rhode PR, Campbell JL. Purification and characterization of proteins that bind to yeast ARSs. J Biol Chem. 1988 Nov 25;263(33):17270–17277. [PubMed]
  • Francesconi SC, Eisenberg S. Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences. Mol Cell Biol. 1989 Jul;9(7):2906–2913. [PMC free article] [PubMed]
  • Diffley JF, Stillman B. Purification of a yeast protein that binds to origins of DNA replication and a transcriptional silencer. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2120–2124. [PubMed]
  • Buchman AR, Kimmerly WJ, Rine J, Kornberg RD. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jan;8(1):210–225. [PMC free article] [PubMed]
  • Walker SS, Francesconi SC, Eisenberg S. A DNA replication enhancer in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4665–4669. [PubMed]
  • Bell SP, Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. [PubMed]
  • Estes HG, Robinson BS, Eisenberg S. At least three distinct proteins are necessary for the reconstitution of a specific multiprotein complex at a eukaryotic chromosomal origin of replication. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11156–11160. [PubMed]
  • Tye BK. The MCM2-3-5 proteins: are they replication licensing factors? Trends Cell Biol. 1994 May;4(5):160–166. [PubMed]
  • Yan H, Merchant AM, Tye BK. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes Dev. 1993 Nov;7(11):2149–2160. [PubMed]
  • Shakibai N, Kumar V, Eisenberg S. The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11569–11574. [PubMed]
  • Boeke JD, Trueheart J, Natsoulis G, Fink GR. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. [PubMed]
  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. [PubMed]
  • Rao H, Stillman B. The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2224–2228. [PubMed]
  • Pellman D, Bagget M, Tu YH, Fink GR, Tu H. Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae. J Cell Biol. 1995 Sep;130(6):1373–1385. [PMC free article] [PubMed]
  • Umek RM, Kowalski D. The ease of DNA unwinding as a determinant of initiation at yeast replication origins. Cell. 1988 Feb 26;52(4):559–567. [PubMed]
  • Fox CA, Loo S, Rivier DH, Foss MA, Rine J. A transcriptional silencer as a specialized origin of replication that establishes functional domains of chromatin. Cold Spring Harb Symp Quant Biol. 1993;58:443–455. [PubMed]
  • Wingender E. Recognition of regulatory regions in genomic sequences. J Biotechnol. 1994 Jun 30;35(2-3):273–280. [PubMed]
  • Rao H, Marahrens Y, Stillman B. Functional conservation of multiple elements in yeast chromosomal replicators. Mol Cell Biol. 1994 Nov;14(11):7643–7651. [PMC free article] [PubMed]
  • DePamphili ML. How transcription factors regulate origins of DNA replication in eukaryotic cells. Trends Cell Biol. 1993 May;3(5):161–167. [PubMed]
  • Ferguson MK, Botchan MR. Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication. J Virol. 1996 Jul;70(7):4193–4199. [PMC free article] [PubMed]
  • Learn B, Karzai AW, McMacken R. Transcription stimulates the establishment of bidirectional lambda DNA replication in vitro. Cold Spring Harb Symp Quant Biol. 1993;58:389–402. [PubMed]
  • Wiltshire S, Raychaudhuri S, Eisenberg S. An Abf1p C-terminal region lacking transcriptional activation potential stimulates a yeast origin of replication. Nucleic Acids Res. 1997 Nov 1;25(21):4250–4256. [PMC free article] [PubMed]
  • Walker SS, Francesconi SC, Tye BK, Eisenberg S. The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):2914–2921. [PMC free article] [PubMed]
  • McNally FJ, Rine J. A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Nov;11(11):5648–5659. [PMC free article] [PubMed]
  • Giniger E, Varnum SM, Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. [PubMed]

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