Stalled replication forks need to be swiftly detected, protected from collapse, and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are ...recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3'-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA, and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.
Abstract
HP1 (heterochromatin protein 1), a key factor for the formation of heterochromatin, binds to the methylated lysine 9 of histone H3 (H3K9me) and represses transcription. While the H3K9me mark ...and HP1 binding are thought to be faithfully propagated to daughter cells, the heterochromatin structure could be dynamically regulated during cell cycle. As evidenced by the well-known phenomenon called position effect variegation (PEV), heterochromatin structure is dynamically and stochastically altered during developmental processes, and thus the expression of genes within or in the vicinity of heterochromatin could be affected by mutations in factors regulating DNA replication as well as by other epigenetic factors. Recent reports show that HP1 also plays an important role in the maintenance and transmission of chromosomes. Like many other factors ensuring faithful chromosome segregation, HP1 family proteins are subjected to posttranslational modifications, most notably phosphorylation, in a cell cycle-dependent manner. Recent studies identified a conserved phosphorylation site that profoundly affects the functions of HP1 during mitotic phase. In this commentary, we discuss dynamic regulation of HP1 protein by phosphorylation during transcriptional repression and cell cycle.
Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication ...fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.
Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more ...complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases.
Display omitted
► Replicator-initiator interactions in prokaryotes are highly specific and efficient. ► Eukaryotic DNA replication is regulated in both “strict" and “soft” manners. ► Rif1 plays a major role in determining spatial and temporal origin firing program. ► Stalled replication fork is swiftly detected/ corrected to avoid genome instability. ► PriA plays a crucial role in detection and restoration of stalled forks in bacteria.
Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA ...replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.
DNA replication is spatially and temporally regulated during S‐phase. DNA replication timing is established in early‐G1‐phase at a point referred to as timing decision point. However, how the ...genome‐wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1‐interacting‐factor‐1), originally identified as a telomere‐binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid‐S replication foci profiles, stimulation of initiation events in early‐S‐phase and changes in long‐range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear‐insoluble structures at late‐M‐to‐early‐G1 and regulates chromatin‐loop sizes. Furthermore, Rif1 colocalizes specifically with the mid‐S replication foci. Thus, Rif1 establishes the mid‐S replication domains that are restrained from being activated at early‐S‐phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher‐order chromatin architecture.
The homologue of the yeast telomeric protein Rif1 regulates the complex temporal programme of human genome replication, possibly controlling chromatin domain establishment at the G1 ‘timing decision point’
DNA replication is central to cell proliferation. Studies in the past six decades since the proposal of a semiconservative mode of DNA replication have confirmed the high degree of conservation of ...the basic machinery of DNA replication from prokaryotes to eukaryotes. However, the need for replication of a substantially longer segment of DNA in coordination with various internal and external signals in eukaryotic cells has led to more complex and versatile regulatory strategies. The replication program in higher eukaryotes is under a dynamic and plastic regulation within a single cell, or within the cell population, or during development. We review here various regulatory mechanisms that control the replication program in eukaryotes and discuss future directions in this dynamic field.
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 rif1Δ, 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 rif1Δ 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 rif1Δ. 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 rif1Δ. 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.
DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing ...and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.
PDF
