Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae.
ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.
Sharma
K
K
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263-0001, USA.
Weinberger
M
M
Huberman
J A
JA
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D002875:Chromosomes / Q000235:genetics / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000378:metabolism; D005838:Genotype; D047908:Intracellular Signaling Peptides and Proteins; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae