The overall objective of this project is to understand in detail how a eukaryotic chromosome replicates and segregates. To this end a 185 kilobase (kb) circular derivative of yeast chromosome III has been cloned and a restriction map constructed. Since the average spacing of replication origins, measured by electron microscopy of replicating chromsomal DNA, is 36 kb, this chromosome is expected to have 5-7 origins of replication. This chromosome and derivatives of it will be used to study the location, structure and efficiency of replication origins, to study the temporal sequence of replication, and to identify genes whose products are necessary for the replication of this chromosome. This particular proposal is addressed to the following issues. Sequences on chromosome III which are good candidates for replication origins have been cloned and mapped. These autonomously replicating sequences (ARS's) allow the extrachromosomal replication of plasmids in yeast. These sequences will be studied to determine whether replication initiates at these sites in vivo, whether these sequences are necessary for normal chromosome function and what DNA sequences are essential for ARS function. Genes whose products interact with ARS's will be identified using a novel selection scheme. Cloned fragments of chromosome III will be used in several experiments designed to determine whether there is a fixed temporal order of replication on the chromosome. If so, then sequences in which the information that specifies the temporal order will be identified. These studies should greatly increase our understanding of a fundamental cellular process, DNA replication. These questions cannot be approached in higher eukaryotes at the present time because their chromosomal DNA's are too large to isolate without breakage and because no autonomously replicating chromosomal sequences have been identified which function in the cells from which they were isolated. Knowledge we gain from the yeast system should be applicable to higher cells, and may help lead to an understanding of the basis of unscheduled DNA synthesis, which is characteristic of malignant cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035679-03
Application #
3288698
Study Section
Genetics Study Section (GEN)
Project Start
1985-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
07107
Irene, Carmela; Theis, James F; Gresham, David et al. (2016) Hst3p, a histone deacetylase, promotes maintenance of Saccharomyces cerevisiae chromosome III lacking efficient replication origins. Mol Genet Genomics 291:271-83
Theis, James F; Irene, Carmela; Dershowitz, Ann et al. (2010) The DNA damage response pathway contributes to the stability of chromosome III derivatives lacking efficient replicators. PLoS Genet 6:e1001227
Chang, Fujung; Theis, James F; Miller, Jeremy et al. (2008) Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site. Mol Cell Biol 28:5071-81
Caldwell, Julie M; Chen, Yinhuai; Schollaert, Kaila L et al. (2008) Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins. J Cell Biol 180:1073-86
Theis, James F; Dershowitz, Ann; Irene, Carmela et al. (2007) Identification of mutations that decrease the stability of a fragment of Saccharomyces cerevisiae chromosome III lacking efficient replicators. Genetics 177:1445-58
Dershowitz, Ann; Snyder, Marylynn; Sbia, Mohammed et al. (2007) Linear derivatives of Saccharomyces cerevisiae chromosome III can be maintained in the absence of autonomously replicating sequence elements. Mol Cell Biol 27:4652-63
Newlon, Carol S; Theis, James F (2002) DNA replication joins the revolution: whole-genome views of DNA replication in budding yeast. Bioessays 24:300-4
Fabiani, L; Irene, C; Aragona, M et al. (2001) A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1. Mol Genet Genomics 266:326-35
Poloumienko, A; Dershowitz, A; De, J et al. (2001) Completion of replication map of Saccharomyces cerevisiae chromosome III. Mol Biol Cell 12:3317-27
Malkova, A; Signon, L; Schaefer, C B et al. (2001) RAD51-independent break-induced replication to repair a broken chromosome depends on a distant enhancer site. Genes Dev 15:1055-60

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