ANALYSIS OF THE TEMPORAL PROGRAM OF REPLICATION INITIATION IN YEAST CHROMOSOMES

The multiple origins of eukaryotic chromosomes vary in the time of the ir initiation during S phase. In the chromosomes of Saccharomyces cere visiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an i nternal position on chromosome XIV. Four origins, spanning similar to 140 kb, initiate replication In the second half of S phase. At least t wo of these internal origins maintain their late replication time on c ircular plasmids. Each of these origins can be separated into two func tional elements: those sequences that provide origin function and thos e that impose late activation, Because the assay for determining repli cation time is costly and laborious, it has not been possible to analy ze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits th e expression of the Escherichia coli dam methylase in yeast and the ch aracteristic period of hemimethylation that transiently follows the pa ssage of a replication fork. The second uses quantitative hybridizatio n to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequen ce by site-specific pop-out recombination. This non-replicating fragme nt acts as an internal control for copy number within and between samp les. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradi ents to detect replicated DNA. With these techniques it should be poss ible to identify the sequences responsible for late initiation, to sea rch for other late replicating regions in the genome, and to begin to analyze the effect that altering the temporal program has on chromosom e function.