GEMC1 is a novel TopBP1-interacting protein involved in chromosomal DNA replication

Chromosomal DNA must be precisely replicated in each cell cycle in order to ensure maintenance of genome stability. Most of the factors controlling this process have been identified in lower eukaryotes. Several factors involved in DNA replication are also important for the cellular response to stress conditions. However, the regulation of DNA replication in multi-cellular organisms is still poorly understood. Using the Xenopus laevis egg cell-free system, we have recently identified a novel vertebrate protein named GEMC1 required for DNA replication. xGEMC1 is a cyclin-dependent kinase (CDK) target required for the Cdc45 loading onto chromatin and it interacts with the checkpoint and replication factor TopBP1, which promotes its binding to chromatin during pre-replication complex formation. Here we discuss our recent findings and propose possible roles for GEMC1. Interestingly, recent studies have identified other proteins with analogous functions, showing a higher level of complexity in metazoan replication control compared to lower eukaryotes.Key words: DNA replication, GEMC1, Sld3, CDK, TopBP1, checkpoint     

GEMC1 is a critical regulator of multiciliated cell differentiation

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1‐deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.     

Tipin/Tim1/And1 protein complex promotes Polα chromatin binding and sister chromatid cohesion

The Tipin/Tim1 complex plays an important role in the S‐phase checkpoint and replication fork stability. However, the biochemical function of this complex is poorly understood. Using Xenopus laevis egg extract we show that Tipin is required for DNA replication in the presence of limiting amount of replication origins. Under these conditions the DNA replication defect correlates with decreased levels of DNA Polα on chromatin. We identified And1, a Polα chromatin‐loading factor, as new Tipin‐binding partner. We found that both Tipin and And1 promote stable binding of Polα to chromatin and that this is required for DNA replication under unchallenged conditions. Strikingly, extracts lacking Tipin and And1 also show reduced sister chromatids cohesion. These data indicate that Tipin/Tim1/And1 form a complex that links stabilization of replication fork and establishment of sister chromatid cohesion.     

Histone Synthesis inTrypanosoma cruzi

Trypanosoma cruziis an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence ofTrypanosomahistones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence ofT. cruzihistones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.     

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.     

Noc3p,a bHLH protein,plays an integral role in the initiation of DNA replication in budding yeast

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.     

Roles of chromatin assembly factor 1 in the epigenetic control of chromatin plasticity

Genetic information embedded in DNA sequence and the epigenetic information marked by modifications on DNA and histones are essential for the life of eukaryotes. Cells have evolved mechanisms of DNA duplication and chromatin restoration to ensure the inheritance of genetic and epigenetic information during cell division and development. In this review, we focus on the maintenance of epigenetic landscape during chromatin dynamics which requires the orchestration of histones and their chaperones. We discuss how epigenetic marks are re-established in the assembly of new chromatin after DNA replication and repair, highlighting the roles of CAF-1 in the process of changing chromatin state. The functions of CAF-1 provide a link between chromatin assembly and epigenetic restoration.     

Xenopus CDC7/DRF1 complex is required for the initiation of DNA replication

The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.     

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.     

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein–protein and protein–DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis‐acting sequences that serve as replication origins and the trans‐acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed. J. Cell. Biochem. 106: 512–520, 2009. © 2009 Wiley‐Liss, Inc.     

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication

During late mitosis and early G₁, a series of proteins are assembled onto replication origins, resulting in them becoming ‘licensed’ for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.     

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.     

Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts

A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.     

The Double Life of UPF1 in RNA and DNA Stability Pathways

The DNA and RNA helicase UPF1 is well known for its central role in Nonsense Mediated RNA Decay (NMD), which promotes degradation of mRNAs containing premature stop codons. However, we have recently demonstrated that human UPF1 is also essential for DNA replication and S phase progression. This function appears to be independent of NMD, which is not required for cell cycle progression. UPF1 physically interacts with the replicative DNA polymerase δ and it associates with chromatin during S phase and upon DNA damage in an ATR-dependent manner. Intriguingly, the human NMD kinase SMG1 is also involved in genome stability pathways and the human NMD-factor EST1A/SMG6 is telomerase-associated and has been implicated in telomere maintenance. Here we review the recent findings, which uncovered the direct roles of UPF1 and other NMD-factors in DNA replication and genome maintenance pathways and suggest functional connections between RNA and DNA metabolism.     

Involvement of fission yeast Clr6-HDAC in regulation of the checkpoint kinase Cds1

Modification of the N-terminal tail of histones is required for various nuclear processes. Here, we show that fission yeast Clr6-HDAC (histone deacetylase) regulates the checkpoint kinase Cds1 when DNA replication encounters a stressful condition. We found that the global level of acetylation of histone H4 was constant throughout the normal cell cycle, but was reduced significantly when the cell recovered from the HU-induced cell cycle arrest (or slow DNA replication). We identified the Clr6-HDAC as a component responsible for the reduction in the level of the H4 acetylation. Although DNA replication was completed, the HU-induced cell cycle arrest could not be released even after removal of HU in the clr6-1 mutant. Under this experimental condition, Cds1 kinase was maintained active and remained bound tightly to chromatin. We also demonstrated that Cds1 was active even after treatment with caffeine, an inhibitor for ATM/ATR that is an activator of Cds1. These results indicate that inactivation of Cds1 requires functional Clr6-HDAC independently of the conventional DNA replication checkpoint. When DNA replication is impeded, Clr6-HDAC activity may monitor damage on chromatin structure/environment, which is required for inactivation of Cds1.