One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1D, a null mutant of rif1 + , a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1D in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1D. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1D. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.

No abstract

No abstract

No abstract

How early-and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1⌬ cells (and in swi1⌬ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1⌬ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein.Initiation of eukaryotic DNA replication proceeds in two steps: assembly of prereplicative complexes (pre-RCs) on the chromosomes during early G 1 and activation of selected preRCs during S phase (22,28). Although the components of pre-RCs and the assembly process as well as regulation of their formation are well understood, the processes of firing still remain largely elusive. Especially, the mechanisms of selection of the origins to be fired and regulation of the timing of firing are two critical issues of eukaryotic DNA replication which are now under intensive study. Studies in the budding yeast Saccharomyces cerevisiae indicate the presence of the origins that are fired efficiently in early to mid-S phase and of others that are fired late in S phase (38). Suppression of late-origin firing is regulated by the Mec1-Rad53/Rad3-Cds1 checkpoint pathway in response to replication stress (25,39,42) and by chromatin structures through histone acetylation (2,12,49). These are conserved in other species as well. However, it is not clear whether there is a critical determinant for early replication on eukaryotic chromosomes during the normal course of S phase. It was also reported in the fission yeast Schizosaccharomyces pombe that early recruitment of origin recognition complex (ORC) and pre-RC components correlates with early firing in S phase (52).Studies in the fission yeast or mammalian cells indicate a great deal of flexibility in origin selection, and it was even proposed that origins are selected from the preformed preRCs in a stochastic manner (6, 36). Indeed, single-cell analyses in yeast cells showed that the site selection for firing occurs rather randomly in each cell cycle of a single cell (36). A reverse correlation between the fork progression rate and firing frequency was reported, and this could also be explained by stochastic activation of pre-RCs (5, 26). On the other hand, genome-wide determination of origins that are fired in the presence of hydroxyurea (HU) clearly indicates the presence of specif...

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1(Chk2/Rad53) through the Rad3(ATR/Mec1)-Mrc1(Claspin) pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.

Short-title: Epigenetic noise and cell identity loss during agingOne Sentence Summary: The act of repairing DNA breaks induces chromatin reorganization and a loss of cell identity that may contribute to mammalian aging 1 SUMMARY All living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.2

A number of different genetic backgrounds and growth conditions bypass DNA replication defects caused by the absence of yeast Hsk1 kinase, demonstrating the plasticity of the eukaryotic DNA replication program.