The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase a that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase a. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.

Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases ␣ and and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.[Keywords: DNA replication forks; Mrc1; Tof1; Csm3; checkpoint; recombination] Supplemental material is available at http://www.genesdev.org.

The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1. INTRODUCTIONEukaryotic cells regulate the progression of DNA replication forks in a highly complex manner to preserve genome stability. For example, cells activate checkpoint kinases in response to defects in DNA synthesis or after the encounter of forks with sites of DNA damage, and these kinases play a key role in preserving the integrity of the fork under such conditions (Andreassen et al., 2006;Branzei and Foiani, 2006). The progression of DNA replication forks is also regulated in diverse ways during the normal process of chromosome replication, and this allows eukaryotic cells to couple chromosome replication to other cellular processes. For example, pausing of a DNA replication fork at a specific site in the mating type region of fission yeast is linked to the establishment of a genomic imprint (Dalgaard and Klar, 2000). In addition, the progression of replication forks is coupled to the establishment of cohesion between sister chromatids, which allows accurate segregation of the chromosomes during mitosis and also facilitates the repair of double-strand breaks in DNA by homologous recombination (Uhlmann and Nasmyth, 1998;Klein et al., 1999;Hanna et al., 2001;Sjogren and Nasmyth, 2001;Cortes-Ledesma and Aguilera, 2006).The progression of eukaryotic DNA replication forks is driven by the heterohexameric DNA helicase MCM2-7 (hereafter MCM), which unwinds the parental DNA duplex and thus allows the replisome machinery to advance (Ishimi, 1997;Labib et al., 2000;Pacek and Walter, 2004;Shechter et al., 2004;Pacek et al., 2006). Work with budding yeast has shown that MCM associates with a large but specific set of regulatory proteins at forks to form so-called "replisome progression complexes" (RPCs) (Gambus et al., 2006). In addition to MCM, other components of RPCs include factors such as Cdc45 and the four GINS proteins that are also required for fork progression, and which may play a role in the activation of MCM Kanemaki et al., 2003;Pacek and Walter, 2004;Pacek et al., 2006). Consistent with this view, a complex of Cdc45-MCM-GINS has been isolated from extracts of Drosophila embryos, and was found to have an associated DNA helicase activity in vitro (Moyer et al., 2006). Moreover, the...

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of prereplicative complexes that are a prerequisite for firing origins during DNA replication in the S phase. In budding yeast, the presence of Cdc6 protein is normally restricted to the G 1 phase of the cell cycle, at least partly because of its proteolytic degradation in the late G 1 /early S phase. Here we show that a Cdc28-dependent mechanism targets p57 CDC6 for degradation in mitotic-arrested budding yeast cells. Consistent with this observation, Cdc6 -7 and Cdc6 -8 proteins, mutants lacking Cdc28 phosphorylation sites, are stabilized relative to wild-type Cdc6. Our data also suggest a correlation between the absence of Cdc28/Clb kinase activity and Cdc6 protein stabilization, because a drop in Cdc28/Clb-associated kinase activity allows mitotic-arrested cells to accumulate Cdc6 protein. Finally, we also show that cdc28 temperature-sensitive G 1 mutants accumulate Cdc6 protein because of a post-transcriptional mechanism. Our data suggest that budding yeast cells target Cdc6 for degradation through a Cdc28-dependent mechanism in each cell cycle.

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of pre-replicative complexes that are required for firing DNA replication at origins at the beginning of S phase. Cdc6p protein levels oscillate during the cell cycle. In a normal cell cycle the presence of this protein is restricted to G 1 , partly because the CDC6 gene is transcribed only during G 1 and partly because the Cdc6p protein is rapidly degraded at late G 1 /early S phase. We report here that the Cdc6p protein is degraded in a Cdc4-dependent manner, suggesting that phosphorylated Cdc6 is specifically recognized by the ubiquitin-mediated proteolysis machinery. Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism. Our data, together with previous observations regarding Cdc6 stability, suggest that under physiological conditions budding yeast cells degrade ubiquitinated Cdc6 every cell cycle at the beginning of S phase.

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.