A region of the Sir1 protein dedicated to recognition of a silencer and required for interaction with the Orc1 protein in saccharomyces cerevisiae.

Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.

[1]  Ellson Y. Chen,et al.  Overview of manual and automated DNA sequencing by the dideoxy chain termination method , 1991 .

[2]  M. Botchan,et al.  Separation of Origin Recognition Complex Functions by Cross-Species Complementation , 1995, Science.

[3]  J. Rine,et al.  Roles of two DNA‐binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer. , 1988, The EMBO journal.

[4]  S. Bell,et al.  Yeast origin recognition complex functions in transcription silencing and DNA replication. , 1993, Science.

[5]  Oscar M. Aparicio,et al.  Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae , 1991, Cell.

[6]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[7]  F. Sherman.  Getting started with yeast. , 1991, Methods in enzymology.

[8]  J. Rine,et al.  Silencing and heritable domains of gene expression. , 1995, Annual review of cell and developmental biology.

[9]  Jasper Rine,et al.  Epigenetic inheritance of transcriptional states in S. cerevisiae , 1989, Cell.

[10]  A. Hinnebusch,et al.  The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7 * , 1997, The Journal of Biological Chemistry.

[11]  J. Rine,et al.  A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[12]  J. Rine,et al.  Silencers and domains of generalized repression. , 1994, Science.

[13]  R. Parker,et al.  A rapid method for localized mutagenesis of yeast genes , 1992, Yeast.

[14]  J. Rine,et al.  The origin recognition complex, SIR1, and the S phase requirement for silencing. , 1997, Science.

[15]  J. Rine,et al.  Yeast spore germination: a requirement for Ras protein activity during re‐entry into the cell cycle , 1997, The EMBO journal.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  E. Nimmo,et al.  Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. , 1996, Journal of cell science.

[18]  R. Sternglanz,et al.  Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing , 1996, Nature.

[19]  J. Rine,et al.  The origin recognition complex has essential functions in transcriptional silencing and chromosomal replication. , 1995, Genes & development.

[20]  David Shore,et al.  Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast , 1993, Cell.

[21]  D. Shore,et al.  RAP1: a protean regulator in yeast. , 1994, Trends in genetics : TIG.

[22]  D. Edmondson,et al.  Chromatin and transcription , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  J. Rine,et al.  The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function. , 1997, Genetics.

[24]  Bruce Stillman,et al.  ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex , 1992, Nature.

[25]  B. Stillman,et al.  A yeast chromosomal origin of DNA replication defined by multiple functional elements. , 1992, Science.

[26]  Rodney Rothstein,et al.  Elevated recombination rates in transcriptionally active DNA , 1989, Cell.

[27]  A. Wolffe,et al.  Deviant nucleosomes: the functional specialization of chromatin. , 1996, Trends in genetics : TIG.

[28]  J. Hicks,et al.  Cloning and characterization of four SIR genes of Saccharomyces cerevisiae , 1986, Molecular and cellular biology.

[29]  A. Hyman,et al.  Structure and function of kinetochores in budding yeast. , 1995, Annual review of cell and developmental biology.

[30]  A. Vershon,et al.  Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene , 1996, Molecular and cellular biology.

[31]  Bruce Stillman,et al.  ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome , 1995, Cell.

[32]  J. Rine,et al.  Sir- and silencer-independent disruption of silencing in Saccharomyces by Sas10p. , 1998, Genetics.

[33]  M. Holland,et al.  Binding Sites for Abundant Nuclear Factors Modulate RNA Polymerase I-dependent Enhancer Function in Saccharomyces cerevisiae(*) , 1995, The Journal of Biological Chemistry.

[34]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[35]  B. Bainbridge,et al.  Genetics , 1981, Experientia.

[36]  M. Yanagida,et al.  Mis6, a Fission Yeast Inner Centromere Protein, Acts during G1/S and Forms Specialized Chromatin Required for Equal Segregation , 1997, Cell.

[37]  H. Kowarzyk.  Structure and Function. , 1910, Nature.

[38]  K. Nasmyth,et al.  A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation , 1987, Cell.

[39]  I. Herskowitz,et al.  Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. , 1987, Genetics.

[40]  S. Elgin,et al.  Heterochromatin and gene regulation in Drosophila. , 1996, Current opinion in genetics & development.