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L L Breeden

Publications and source records attributed to L L Breeden.

9 recordsLinked to original sources
PubMed2001

Early cell cycle box-mediated transcription of CLN3 and SWI4 contributes to the proper timing of the G(1)-to-S transition in budding yeast.

The Cln3-Cdc28 kinase is required to activate the Swi4-Swi6 transcription complex which induces CLN1 and CLN2 transcription in late G(1) and drives the transition to S. Cln3 and Swi4 are both rate limiting for G(1) progression, and they are coordinately transcribed to peak at the M/G(1) boundary. Early cell cycle box (ECB) elements, which confer M/G(1)-specific transcription, have been found in both promoters, and elimination of all ECB elements from the CLN3 promoter causes both a loss of periodicity and Cln3-deficient phenotypes, which include an extended G(1) interval and increased cell volume. Mutants lacking the ECB elements in both the CLN3 and SWI4 promoters have low and deregulated levels of CLN transcripts, and the G(1)-to-S transition for these mutants is delayed and highly variable. These observations support the view that the coordinated rise of Cln3 and Swi4 levels mediated by ECB-dependent transcription controls the timing of the G(1)-to-S phase transition.

Blotting, Northern↗
PubMed2000

Cyclin transcription: Timing is everything.

The stage-specific activation of cyclin-dependent kinases controls the order and timing of cell-cycle transitions. Recent studies offer insight into the mechanism of cell-cycle-regulated transcription of the mitotic cyclins of budding yeast.

Cell Cycle↗
PubMed1998

Mutation and modeling analysis of the Saccharomyces cerevisiae Swi6 ankyrin repeats.

The Swi4-Swi6 family of transcription factors confers G1/S specific transcription in budding and fission yeast. These proteins contain four ankyrin repeats, which are present in a large number of functionally diverse proteins and have been shown to be important for protein-protein interaction. However, no specific sequence has been identified that is diagnostic of an ankyrin repeat-interacting protein. To determine the function of the ankyrin repeats of Swi6, we generated both random and site-directed mutations within the ankyrin repeat domain of Swi6 and assayed the transcriptional function of these mutant swi6 alleles. We found six single mutations, scattered within the first and the fourth repeats, that generate a temperature-sensitive Swi6 protein. In addition, we found that alanine substitutions for the most conserved residues in each repeat were highly deleterious and also confer temperature sensitivity. Most of these swi6 alleles are able to form ternary complexes with Swi4 and DNA, but these complexes display reduced mobility in band-shift gels, suggesting a dramatic conformational change. We have modeled the ankyrin repeats of Swi6 using the coordinates derived for 53BP2 and find that, despite its low level of sequence conservation, these modeling studies and our mutation data are consistent with Swi6 having a structure very similar to that of 53BP2. Moreover, all but one of our single mutants and all of the site-directed mutants disrupt critical structural features of the predicted folding pattern of these repeats. We conclude that the ankyrin repeats play a major structural role in Swi6. Ankyrin repeats are unlikely to have inherent protein or DNA binding properties. However, they form a characteristic and stable structure with surfaces that may be tailored for many different macromolecular interactions.

Alanine↗
PubMed1997

Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 and CLN2 transcription occur in response to DNA damage in Saccharomyces cerevisiae.

Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage. We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1. This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to alpha-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells. We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels. The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well. In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6. This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro. On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription.

Alkylating Agents↗
PubMed1997

A novel Mcm1-dependent element in the SWI4, CLN3, CDC6, and CDC47 promoters activates M/G1-specific transcription.

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication.

Cell Cycle Proteins↗
PubMed1997

Cell cycle-dependent transcription of CLN1 involves swi4 binding to MCB-like elements.

Two promoter elements have been defined that activate G1/S-specific transcription in Saccharomyces cerevisiae. SCB elements (CACGAAA) are activated by the Swi4-Swi6 complex, and MCB elements (ACGCGTNA) are activated by the Mbp1-Swi6 complex. CLN1 encodes a cyclin which is expressed during this interval, and requires Swi4 and Swi6 for peak transcription, but it has no consensus SCB elements in its promoter. Two SCB-like sequences had been previously noted and suggested to be the functional promoter elements. Our studies indicate that these sequences are unable to activate transcription of a lacZ reporter construct, or to bind Swi4-Swi6 complexes in vitro. However, a cluster of three sequences resembling MCB sequences are active promoter elements, sufficient to confer G1/S-specific transcription to a reporter. These sites are the predominant activation elements in the CLN1 promoter, and despite their resemblance to MCB elements, they bind Swi4-Swi6 complexes in vitro and require Swi4 and Swi6 for their activity in vivo. This indicates that the sequences that promote Swi4/Swi6 binding have not been fully defined, or that there are multiple Swi4- and Swi6-containing complexes with distinct DNA binding specificities. In addition to these novel Swi4/Swi6-binding sites, these studies also show that there must be at least one novel promoter element that can confer G1/S-specific transcription to CLN1, because when all the potential SCB- and MCB-like sequences are eliminated the transcript is still cell cycle regulated.

Cell Cycle↗
PubMed1996

A new Saccharomyces cerevisiae ankyrin repeat-encoding gene required for a normal rate of cell proliferation.

We have characterized a new ankyrin (ANK) repeat-containing Saccharomyces cerevisiae gene, YAR1, located between the HSP82 and SUI3 genes on chromosome XVI. YAR1 encodes a 200-amino-acid (aa) protein with two ANK repeat motifs and an acidic C terminus rich in PEST-like sequences. The Yar1 ANK repeats are most similar to the conserved ANK repeats in the yeast cell cycle transcription factor, Swi6. We show that YAR1 is transcribed as an 800-nucleotide (nt) poly(A)+ mRNA from a promoter lacking a consensus TATA sequence. YAR1 is transcribed in both haploid and diploid cells, and in haploid cells arrested in G1 with alpha-factor or in S phase with hydroxyurea. YAR1 shares an intergenic region with HSP82, and while HSP82 transcription is induced 15-fold by heat shock, transcription of YAR1 is transiently repressed by heat shock. We show that YAR1 is not an essential gene, but that haploid cells bearing a yar1 deletion grow significantly more slowly than do isogenic wild-type cells, especially at low temperature.

Amino Acid Sequence↗
PubMed1995

Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localization.

The Swi6 transcription factor, required for G1/S-specific gene expression in Saccharomyces cerevisiae, is highly phosphorylated in vivo. Within the limits of resolution of the peptide analysis, the synchrony, and the time intervals tested, serine 160 appears to be the only site of phosphorylation in Swi6 that varies during the cell cycle. Serine 160 resides within a Cdc28 consensus phosphorylation site and its phosphorylation occurs at about the time of maximal transcription of Swi6- and Cdc28-dependent genes containing SCB or MCB elements. However, phosphorylation at this site is not Cdc28-dependent, nor does it control G1/S-specific transcription. The role of the cell cycle-regulated phosphorylation is to control the subcellular localization of Swi6. Phosphorylation of serine 160 persists from late G1 until late M phase, and Swi6 is predominantly cytoplasmic during this time. Aspartate substitution for serine 160 inhibits nuclear localization throughout the cycle. Swi6 enters the nucleus late in M phase and throughout G1, when serine 160 is hypophosphorylated. Alanine substitution at position 160 allows nuclear entry of Swi6 throughout the cell cycle. GFP fusions with the N-terminal one-third of Swi6 display the same cell cycle-regulated localization as Swi6.

Amino Acid Sequence↗