Bonita J. Brewer

The localization of replication origins on ARS plasmids in S. cerevisiae

Replication intermediates from the yeast 2 microns plasmid and a recombinant plasmid containing the yeast autonomous replication sequence ARS1 have been analyzed by two-dimensional agarose gel electrophoresis. Plasmid replication proceeds through theta-shaped (Cairns) intermediates, terminating in multiply interlocked catenanes that are resolved during S phase to monomer plasmids. Restriction fragments derived from the Cairns forms contain replication forks and bubbles that behave differently from one another when subjected to high voltage and agarose concentrations. The two-dimensional gel patterns observed for different restriction fragments from these two plasmids indicate that in each plasmid there is a single, specific origin of replication that maps, within the limits of our resolution, to the ARS element. Our results strongly support the long-standing assumption that in Saccharomyces cerevisiae an ARS is an origin of replication.

Histone Acetylation Regulates the Time of Replication Origin Firing

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

Analysis of replication intermediates by two-dimensional agarose gel electrophoresis

Publisher Summary This chapter focuses on the analysis of replication intermediates by two-dimensional (2D) agarose gel electrophoresis. The first step in the analysis of replication intermediates on 2-D gels involves the isolation of branched molecules in a way that preserves their structure. Branched molecules are delicate, and shearing is minimized by use of large-bore pipette tips and gentle hand mixing. Shearing can lead to loss of replication intermediates, as well as generation of novel branched artifacts. Exposure of the DNA to excessive heat is avoided in order to preserve the integrity of DNA at the fork. Restriction fragments of larger or smaller size can be analyzed on 2-D gels if some modifications are made to the electrophoresis conditions. Finding the correct electrophoresis conditions for larger or smaller fragments require some experimentation using fragments of known size and replication pattern. Fragments that are larger than 5.5 to 6 kb must be run under conditions of lower agarose concentration and lower voltage in both dimensions in order to separate successfully the bubble and simple Y arcs. If gels are run under the conditions intended for smaller fragments (2 to 5.5 kb), the bubble and simple Y patterns are not clearly separated, and the signals for each are distorted. After the second dimension of electrophoresis, nonreplicating molecules are seen as a shallow arc, whereas the rare replicating molecules run above this arc.

Replication in Hydroxyurea: It's a Matter of Time

ABSTRACT Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the “intra-S phase checkpoint.” Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

CLB5-Dependent Activation of Late Replication Origins in S. cerevisiae

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

A role for recombination junctions in the segregation of mitochondrial DNA in yeast

In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

A yeast origin of replication is activated late in S phase

The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.

Replication profile of Saccharomyces cerevisiae chromosome VI

An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae.

A question of time: Replication origins of eukaryotic chromosomes

Walton L. Fangman and Bonita J. Brewer Department of Genetics SK-50 University of Washington Seattle, Washington 98195 Each chromosome of a eukaryotic organism contains many replication origins (Figure 1) from which DNA is effi- ciently duplicated during S phase of the cell cycle. Recent work raises new questions about the nature of these ori- gins and their regulation. What Are Origins? Like initiation of transcription at a promoter, initiation of replication at an origin can be thought of as requiring three discrete steps: the recognition of one or more cis-acting elements by specific initiation proteins, the localized un- winding of the DNA helix, and the selection of a site for the initiation of polymerization. Continuing the analogy to a promoter, it is clear that these three steps do not necessar- ily have to occur at the same site and may differ greatly in their degree of sequence specificity. Assays to detect ori- gin function may detect one or more of these steps of initiation and thus may give different results if the events occur at different sites. Replication origins have been best defined in the yeast Saccharomyces cerevisiae. The autonomous replication sequence (ARS) assay, which detects the cis-acting se- quences required for origin function in plasmids, has al- lowed mutational analysis with increasing resolution (Mar- ahrens and Stillman, 1992). ARS elements consist of only 100-200 bp and include a conserved 11 bp core consen- sus sequence and several other less conserved elements that are required for or enhance the maintenance of a plasmid. Mapping of the site of initiation by two-dimen- sional gels (Brewer and Fangman, 1987; Huberman et al., 1987) reveals that replication initiation on plasmids occurs in the vicinity of the ARS element (within a few hundred base pairs of the ARS consensus). The exact size of the initiation zone and the locations of priming sites for DNA chain elongation are yet to be determined. Two-dimensional gel analysis of replicating chromo- somal DNA fragments has shown that many but not all ARS elements are active as origins in the chromosome, and that initiation is dependent on a functional ARS ele- ment (Dubey et al., 1991; Rivier and Rine, 1992; Desh- pande and Newlon, 1992). Quantitative assessments show that specific origins can have eff iciencies of activation that range from .90 per cell cycle (Dubey et al., 1991; Ferguson et al., 1991). The basis for differences in effi- ciency is not known. Do All Eukaryotes Have Specific Origins? A simple, reliable ARS assay has not been achieved for any eukaryote other than yeast. However, by physical means, examples of specific initiation have been observed in both Physarum and Tetrahymena (Benard and Pierron, 1992; Cech and Brehm, 1981). In these cases, the speci- ficity of initiation can be interpreted as evidence for specific

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.