ORIGIN ACTIVATION AND FORMATION OF SINGLE-STRAND TG1-3 TAILS OCCUR SEQUENTIALLY IN LATE S-PHASE ON A YEAST LINEAR PLASMID

In order to understand the mechanisms leading to the complete duplicat ion of linear eukaryotic chromosomes, the temporal order of the events involved in replication of a 7.5-kb Saccharomyces cerevisiae linear p lasmid called YLpFAT10 was determined. Two-dimensional agarose gel ele ctrophoresis was used to map the position of the replication origin an d the direction of replication fork movement through the plasmid. Repl ication began near the center of ULpFAT10 at the site in the 2 mum seq uences that corresponds to the 2 mum origin of DNA replication. Replic ation forks proceeded bidirectionally from the origin to the ends of Y LpFAT10. Thus, yeast telomeres do not themselves act as origins of DNA replication. The time of origin utilization on YLpFAT10 and on circul ar 2 mum DNA in the same cells was determined both by two-dimensional gel electrophoresis and by density transfer experiments. As expected, 2 mum DNA replicated in early S phase. However, replication of YLpFAT1 0 occurred in late S phase. Thus, the time of activation of the 2 mum origin depended upon its physical context. Density transfer experiment s established that the acquisition of telomeric TG1-3 single-strand ta ils, a predicted intermediate in telomere replication, occurred immedi ately after the replication forks approached the ends of YLpFAT10. Thu s, telomere replication may be the very last step in S phase.