Anatomy and Dynamics of DNA Replication Fork Movement in Yeast Telomeric Regions

Replication of the linear DNA of a eukaryotic chromosome imposes a problem of end replication, as originally predicted by Watson (40) and Olovnikov (31). While the synthesis of the leading strand can proceed to the very end of the template, the lagging strand is predicted to shorten upon every round of replication in each cell cycle. Most eukaryotes solve the end-replication problem by maintaining specific repetitive DNA sequences at their chromosome ends, called telomeres, by the enzyme telomerase, which elongates the 3′ end of the telomeric DNA in a sequence-specific manner. In those rarer situations in which a eukaryote does not have telomerase, other multiple repeats, such as transposable elements in the fruit fly Drosophila melanogaster, are periodically added to their chromosome ends. The yeast Saccharomyces cerevisiae has telomeres containing ∼250 to 350 bp of TG1-3 repeats and uses telomerase for their maintenance. About two-thirds of the 32 telomeres in haploid cells carry one or more copies of subtelomeric Y′ elements (see reference 32 for a review). Members of the major class of Y′ elements are 6.7 kb long, and there is a minor 5.2-kb class; they are always arranged in the same orientation, such that multiple Y′ elements form directly repeating arrays separated by short stretches of telomeric TG1-3 DNA. Replication of eukaryotic chromosomes initiates at autonomously replicating sequence (ARS) elements—origins of replication present at multiple locations on every chromosome. Every Y′ element contains an ARS (4). While many genomic ARSs are fired early in S phase, it has been reported, by using the density transfer method, that Y′ repeats replicate late in S phase (11, 28, 33, 39). Telomeric chromatin has been implicated in determining the timing of activation of subtelomeric ARSs. An ARS placed on a circular plasmid containing telomeric TG1-3 repeats initiates replication early, but if the plasmid is linearized and therefore contains telomeres, the ARS is fired late (11). Furthermore, deletion of Sir3p, one of the components of telomeric heterochromatin, causes the telomeres to replicate in early S phase (39). These results raise the question of whether all the Y′ elements initiate their ARSs synchronously in late S phase or whether the timing of replication is different for the terminal and internal elements, since only the former are positioned next to a terminal telomeric TG1-3 tract. Replication fork movement does not proceed monotonically. Programmed replication fork pausing is conserved from bacteria to higher plants and animals and can contribute to genomic stability. Polar replication fork blocks can ensure unidirectional replication at a certain region of a genome, thereby playing regulatory roles in different cellular processes. For example, in bacteria, polar replication pausing coordinates termination of bidirectional chromosome replication so that the two replication forks meet at the terminus region where newly synthesized chromosomes are to be decatenated (reviewed in reference 34). On either side of the terminus, three sites (Ter) are bound by the protein Tus, and the resultant DNA-protein complex blocks the progression of replication forks in a polar manner. In the mating type locus of the fission yeast Schizosaccharomyces pombe, replication pausing regulates the orientation of DNA replication, thereby determining whether cells undergo mating type switching (9). Many organisms have been shown to have specific replication blocks near ribosomal DNA (rDNA) genes. In the budding yeast S. cerevisiae, replication fork pausing has been found at rDNA and telomeres, and the Rrm3 helicase has been shown to alleviate it at both loci (19, 20). While the replication pausing at rDNA is caused by the DNA-binding protein Fob1 (21, 22), the key determinants that impose the replication barrier at telomeres are unknown. Toward a fuller understanding of the role of replication in telomere maintenance in vivo, here we have analyzed replication of natural yeast telomeric regions, including the subtelomeric Y′ elements, to address the question of the nature and architecture of replication fork pausing at Y′ telomeres.