In this study we have examined the effect of deleting Ku on the S. cerevisiae temporal program of DNA replication. We discovered that Ku can influence replication timing of chromosome ends over a long range, affecting the replication time of sequences up to 80 kb from telomeres. Our genome-wide study showed that the yku70 mutation affects the replication timing of all telomeres to some extent. The effect on some telomeres extends over tens of kilobases, but on others (e.g., XIII-right, XV-right) the effect is restricted to the chromosome end. The extent of the effect on replication timing may reflect the number, configuration, and normal initiation time of replication origins close to each telomere.
One caveat in interpreting microarray-based replication timing data arises from the presence of nonunique sequences within the terminal 20–30 kb of many chromosomes arms (23 of the 32 telomeres falling into groups sharing some degree of homology; Louis, 1995
). Such similarity compromises the uniqueness of some microarray probes, so that the replication timing data for some chromosome ends must be interpreted with caution. We focused on chromosome VI-right in several experiments because it contains unique sequence to within ~500 bp of the chromosome end, permitting reliable probe and primer design.
We tested whether Ku modulates telomere replication dynamics by affecting nucleosome modifications that have previously been implicated in control of replication timing—namely, acetylation of the histone N-terminal tails at residues H4K5, 8, 12, and 16 or H3K18. We did not detect changes in acetylation of these residues at telomere-proximal replication origins that account for the effects of yku70
on replication initiation, suggesting that the advancement in replication time of telomere regions in yku70
is independent of histone tail acetylation. It remains possible that a different chromatin modification might be involved in mediating the effect of Ku on initiation time of telomere-proximal origins (Pryde et al.
Loss of Ku function has several effects on yeast telomere organization that could potentially be related to the replication timing defect. Specifically, deletion of YKU70
causes loss of normal telomere positioning at the nuclear periphery, failure of subtelomeric transcriptional silencing, and abnormally short telomeres (Boulton and Jackson, 1998
; Laroche et al.
; Stellwagen et al.
). Deregulated telomere replication timing in yku70
does not appear to be due to defective telomere subnuclear positioning (because other mutants that derail telomere positioning do not affect replication timing; Hiraga et al.
) or to the telomeric silencing defect (because deleting Sir components has a milder effect than yku70
on telomere replication timing; Stevenson and Gottschling, 1999
; Cosgrove et al.
). We created a pif1 yku70
mutant to examine whether the replication timing defect of cells lacking Ku can be rescued by restored telomere length. We found that a pif1 yku70
strain has telomeres of almost normal length and that they replicate at close to their normal late time in S phase. Because pif1 yku70
mutant cells lack Ku function but have restored telomere length and replication timing, this observation suggests that the effect of the yku70
mutation on telomere replication timing is most likely a consequence of telomere shortening. Consistent with this suggestion, the yku70 elg1
mutant showed slight rescue of telomere length and similarly mild restoration of replication timing.
Our observations are consistent with those from the Shore laboratory demonstrating that an engineered short telomere is reprogrammed to replicate early. Specifically, recombinational excision during G1 of a subtelomeric TG1–3
tract causes early replication of that telomere in the subsequent S phase (Bianchi and Shore, 2007
). The effects of such artificial telomere truncation on replication appear similar to those of the yku70
mutation: In particular, either the yku70
mutation or engineered shortening results in advancement of the replication time of both subtelomeric replication origin sequences (i.e., those within X and Y′ elements) and telomere-proximal origins, such as ARS522
(Cosgrove et al.
; Bianchi and Shore, 2007
How is information on telomere length relayed to telomere-proximal replication origins? To begin to address this question, we tested whether the Rif1 protein, which has a central role in measuring TG1–3 tract length to regulate telomerase activity, is also involved in measuring TG1–3 tract length to control replication timing. Both pif1 and rif1 mutants have elongated telomeres but for completely different reasons: An important difference between these mutants is that the pif1 mutant has intact telomere length–sensing machinery, whereas in a rif1 mutant this machinery is defective. We found that a rif1 strain replicates its telomere-proximal sequences early despite its greatly extended terminal TG1–3 tract length, suggesting that rif1 cells lack the ability to transmit telomere length information to nearby replication origins (as well as failing to repress telomerase activity at long telomeres). illustrates our model for the role of Rif1 in telomere length–controlled replication timing in wild-type and mutant strains. The most likely explanation for the early replication of rif1 telomeres is that, without Rif1 protein, cells lack the machinery required to detect their long telomeres and program the initiation time of nearby replication origins accordingly (). In other words, in the absence of Rif1 the cells constitutively interpret their terminal TG1–3 tracts as being critically short, and so engage the mechanism that causes short telomeres to replicate early.
FIGURE 9: Model of replication timing control by Rif1-mediated telomere length measurement. (A) In wild-type cells, terminal TG1–3 tract length is “counted” by activated Rif1 molecules (gray hexagons). If TG1–3 repeat length is sufficient, (more ...)
Our findings do not address the mechanism by which telomere-associated Rif1 protein transmits information to replication origins that may (like ARS522
) lie tens of kilobases distant from the chromosome end. Our results suggest that the effect of yku70
(and telomere length) on replication timing is not mediated by altered acetylation state of the histone H3 or H4 tails. Although changes in histone tail acetylation clearly can affect replication origin initiation (Vogelauer et al.
), they do not appear to mediate the long-range effect of telomeres on replication timing. In their study of the effects of deacetylase Rpd3 and HAT Gcn5 on replication dynamics, Vogelauer et al.
concentrated on internal late replication domains and did not examine telomere-proximal origins. One possibility therefore is that histone tail acetylation state mediates origin initiation time within internal late replication domains (distant from telomeres), but a different mechanism (not acting through histone tail acetylation) mediates the effect of telomeres on replication timing. We propose that the programming of replication time close to chromosome ends occurs through a Rif1-dependent mechanism in which TG1–3
tract length is measured, and the information is fed into an unidentified pathway that controls replication initiation time. We are investigating the possibility that, as well as measuring TG1–3
tract length, an activated form of Rif1 itself transmits the telomere-length signal to replication origins, regulating replication initiation through an effect on the Cdc7 kinase replication initiation factor. Such a role for S. cerevisiae
Rif1 in replication control could be related to the reported function of the human Rif1 homologue, which is involved in regulating DNA replication of heterochromatic domains (Buonomo et al.
). Understanding the pathways by which yeast telomeres affect the replication program of nearby sequences will elucidate the links between telomere biology and replication and may help uncover general mechanisms that regulate DNA replication.