Replication is central to the biological role of DNA as the molecule of inheritance. The control of DNA replication ensures that chromosomes are duplicated in a timely and precise way. All available evidence indicates that controls operate at the point of initiation of replication at individual origins scattered at high density over eukaryotic chromosomes. It is the control of replication origins that we propose to investigate. Our understanding of these controls is very poor, primarily because replication origins themselves have been elusive. Recently, two sensitive gel electrophesis techniques have been developed for identifying origins in chromosomal DNA. Our studies in the yeast Saccharomyces cerevisiae have revealed that the initiation activity of origins depends to a large extent on their chromosomal context. Three aspects of contextual control of origin use in yeast will be examined. (I) When origins are located in close tandem arrays--in plasmid multimers and in the rDNA locus--many potential origins are not used. What is responsible for the inactivity? Are there spacing constraints? And, how are the active origins chosen? (II) Replication initiation in the transcriptionally active rDNA repeats occurs in the non-transcribed spacer and replication is unidirectional with the active fork moving the transcriptional direction. Does the transcriptional activity of other chromosomal regions influence origin use? Has evolution favored the placement of origins that permits replication forks to follow transcription complexes through actively transcribed genes, rather than colliding head-on with them? (III) Chromosomal origins located near telomeres (the physical ends of chromosomes) are activated later in S phase than origins located at internal sites. What feature of telomeres influences origin activation times, and are some origins inactive because of their proximity to telomeres? Our approach to these questions involves making directed replacements and alterations in yeast plasmids and chromosomes and using our recently developed 2-D gel technique to identify active origins and to estimate the efficiency of their activation. The answers to these questions will lead to a greater understanding of the regulation of chromosome replication in normal cells and in those with altered growth properties, such as cancer cells.
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