DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing. INTRODUCTIONEukaryotic DNA replication begins at discrete origins distributed along chromosomes. Although much progress has been made in understanding the mechanisms that establish and activate individual origins, the manner in which origin firing is coordinately regulated spatially along the chromosomes and temporally throughout S phase remains unclear (Kelly and Brown, 2000;Gilbert, 2001;Schwob, 2004). These questions are of particular interest because in most cases origin firing is not efficient; that is, a particular origin will fire in only a fraction of cell cycles. The observation that origins do not fire in every cell cycle raises the question of how individual origins are selected to fire and if that selection is distributed in a coordinated manner. In the absence of such coordination, origin firing would be randomly distributed, leading to the random gap problem; some cells would have large gaps between origin firing that would take a long time to replicate (Lucas et al., 2000;Herrick et al., 2002;Hyrien et al., 2003;Jun et al., 2004). Simple models of replication kinetics predict that if replication origins are randomly distributed, ϳ5% of the cells will have such large gaps between active origins that they will take four times longer than average to replicate (see Materials and Methods for calculations). Alternatively, cells could have a mechanism that evenly distributes origin firing across the genome; they would thus avoid the random gap problem, and replication would be efficient. Models for such mechanisms have been proposed; for instance, origins within specific clusters could be selected to fire, or active origins could suppress their neighbors by lateral inhibition (Mesner et al., 2003;Shechter and Gautier, 2005). However, little direct evidence exists to either support or refute these models.Origin structure and function has been well characterized in the budding yeast Saccharomyces cerevisiae (Kelly and Brown, 2000;Gilbert, 2001). Budding yeast have small (ϳ100 base pairs) origins characterized by a 17-b...

The structure of the telomeric DNA has been a subject of extensive investigation in recent years due to the realization that it has important functional roles to play in vivo and the observations that truncated telomeric sequences exhibit a great variety of 3D structures in aqueous solutions. In this context, we describe here NMR structural studies on two truncated human telomeric DNA sequences, d-AG(3)T and d-TAG(3)T in solutions containing K(+)ions. The G(3)stretches in both the oligonucleotides were seen to form parallel-stranded quadruplexes. However, the AG(3)segment as a whole, had different structural characteristics. The structure of d-AG(3)T revealed the formation of a novel A-tetrad, which was not seen in d-TAG(3)T. The A's in the tetrad had syn glycosidic conformation as opposed to the anti conformation of the G's in the G-tetrads. The A-tetrad stacked well over the adjacent G-tetrad and the twist angle at this step was smaller in d-AG(3)T than in d-TAG(3)T. These observations are expected to be significant from the point of view of structural diversity and recognition in telomeres.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G 1 /S transcriptional program by directly regulating MBF, the G 1 /S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G 1 /S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G 1 /S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.In response to inhibition of DNA replication, the replication checkpoint arrests the cell cycle, stabilizes replication forks, and regulates transcription (3, 5). The transcriptional branch of the checkpoint response upregulates genes thought to be important for cells to survive prolonged replication arrest and to synthesize extra deoxynucleotides. A commonly used trigger of the replication checkpoint is treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU), which prevents deoxynucleotide synthesis, thus preventing DNA replication. HU treatment activates a conserved checkpoint signaling pathway (3). In the fission yeast Schizosaccharomyces pombe, the central checkpoint kinase Rad3 (the homolog of the budding yeast Mec1 and metazoan ATM and ATR) activates the Sphase checkpoint effector kinase Cds1 (the homolog of the budding yeast Rad53 and Dun1, also called Chk2 in mammals), which regulates the downstream targets of the checkpoint (3). In the budding yeast Saccharomyces cerevisiae, Rad53 and Dun1 are thought to control transcription mainly through the Rfx1/Crt1 transcriptional repressor, which regulates a replication and DNA damage checkpoint-specific transcriptional program (21). However, the Rfx1 regulatory circuit is not evolutionarily conserved and it is unclear how the replication checkpoint regulates transcription in other eukaryotes.During a normal fission yeast cell cycle, G 1 /S transcription is regulated by the MBF transcription factor, also known as DSC1, and its negative regulator Nrm1 (1, 10). MBF-including the proteins Cdc10, Res1, Res2, Rep1, and Rep2-and Nrm1 are conserved in budding yeast, which also contains the paralogou...