Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae.

Abstract

Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements.GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT a genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences within the (Cl_3A)-repeat region at yeast telomeres.Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCAN NCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR 1, and HML I and were associated with ARS1, ARS2, and the 2,um plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DEDI genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.Eucaryotic chromosomes appear to be organized in domains governing such processes as gene expression, DNA replication, and chromosome condensation. In an effort to define the biochemical components directing these events, we have been attracted to the features of the mating type loci of Saccharomyces cerevisiae (reviewed in reference 33). Haploid yeast cells of the a or a mating type express different genes at MAT, in the middle of chromosome III (Fig. 1). The alternate forms of this locus, MATa and MA To, encode distinct regulatory factors al and a2 or al and oa2, but have extensive regions of homology, referred to as W, X, and Z (2, 61). There are, in addition, silent copies of genes specifying each mating type stored on the left and right arms of chromosome III at HMLa and HMRa, respectively. In homothallic strains, the mating type of successive generations switches in a tightly regulated manner (29,35,80). Switching is accomplished by gene conversion whereby sequences at MAT are replaced by those at HML or HMR (33,47). This process is initiated with the cleavage of MAT DNA in Z by a specific endonuclease that is the product of the HO gene (49, 50). The silent copies of the mating type loci are controlled by negative regulatory sequences located over 1,000 base pairs (bp) away from the promoters for these genes. Deletion analysis of HML and HMR has revealed cis-acting regulatory elements, E and I, that flank each locus on the left and right sides, respectivel...

Telomere G-strand structure and function analyzed by chemical protection, base analog substitution, and utilization by telomerase in vitro

“…In fact, proteins that lack telomerase activity but bind to telomeres have been identified in several organisms. Some of these proteins have nontelomeric cellular functions including transcriptional activation (Buchman et al, 1988) and ultrastructural roles (Shoeman et a]., 1988). Others appear to bind specifically to the duplex (Gottschling & Cech, 1984;Gottschling & Zakian, 1986;Berman et al, 1986) while another class bind specifically to the G-strand overhang (Gottschling & Zakian, 1986;Price & Cech, 1987;B.…”

Human telomeres are attached to the nuclear matrix.

“…In Oxytricha and Euplotes, the protruding G-rich repeats are bound by a telomere capping complex which protects telomeres from ligation and exonucleolytic attack in vitro (Lipps et al, 1982;Gottschling and Cech, 1984;Gottsc.hling and Zakian, 1986;Price andCech, 1987, 1989;Raghuraman et al, 1989;Price, 1990). Telomere capping complexes have not been identified in other organisms, but genetic and biochemical experiments suggest that the telomeres of Saccharomyces cerevisiae interact with the repressor/activator protein RAPI (Berman et al, 1986;Buchman et al, 1988;Longtine et al, 1989;Conrad et al, 1990;Lustig et al, 1990). The function of the RAPI -telomere association is not known.…”

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

“…The relative orientation of each match is also shown; the location of the box above or below the line indicates the location of the T-rich strand of the match in the upper or lower strand of the sequence, respectively. These Ars' sequences contain no obvious matches to the ABF-1 binding site as defined by Buchman et al (6).…”