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A foreword to "Cold Spring Harbor Laboratory" is presented.

The article discusses the Symposium on 21st Century Genetics: Genes at Work organized by the Cold Spring Harbor Laboratory in Cold Spring Harbor, New York in 2015.

Nucleotide excision repair (NER) in yeast is effected by the concerted action of a large complex of proteins. Recently, we identified a stable subcomplex containing the yeast Rad7 and Rad16 proteins. Here, we report the identification of autonomously replicating sequence binding factor 1 (ABF1) as a component of the Rad7/Rad16 NER subcomplex. Yeast ABF1 protein is encoded by an essential gene required for DNA replication, transcriptional regulation, and gene silencing. We show that ABF1 plays a direct role in NER in vitro. Additionally, consistent with a role of ABF1 protein in NER in vivo, we show that certain temperature-sensitive abf1 mutant strains that are defective in DNA replication are specifically defective in the removal of photoproducts by NER and are sensitive to killing by ultraviolet (UV) radiation. These studies define a novel and unexpected role for ABF1 protein during NER in yeast. [ABSTRACT FROM AUTHOR]

Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase. [ABSTRACT FROM AUTHOR]

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2- 7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability. [ABSTRACT FROM AUTHOR]

Summary: The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56dim NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients' fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients' growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56dim subset in patients was associated with a lower rate of NK CD56bright cell proliferation, and the maturation of NK CD56bright cells toward an NK CD56dim phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency. [ABSTRACT FROM AUTHOR]

Centrosomes, each containing a pair of centrioles, organize microtubules in animal cells, particularly during mitosis, DNA and centrosomes are normally duplicated once before cell division to maintain optimal genome integrity. We report a new role for the Orc1 protein, a subunit of the origin recognition complex (ORC) that is a key component of the DNA replication licensing machinery, in controlling centriole and centrosome copy number in human cells, independent of its role in DNA replication. Cyclin A promotes Orc1 localization to centrosomes where Orc1 prevents Cyclin E-dependent reduplication of both centrioles and centrosomes in a single cell division cycle. The data suggest that Orc1 is a regulator of centriole and centrosome reduplication as well as the initiation of DNA replication. [ABSTRACT FROM AUTHOR]

Global genome nucleotide excision repair (GG-NER) removes DNA damage from nontranscribing DNA. In Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for GG-NER. We have reported that autonomously replicating sequence-binding factor 1 (ABF1) protein forms a stable complex with Rad7 and Rad16 proteins. ABF1 functions in transcription, replication, gene silencing, and NER in yeast. Here we show that binding of ABF1 to its DNA recognition sequence found at multiple genomic locations promotes efficient GG-NER in yeast. Mutation of the I silencer ABF1-binding site at the HMLα locus caused loss of ABF1 binding, which resulted in a domain of reduced GG-NER efficiency on one side of the ABF1-binding site. During GG-NER, nucleosome positioning at this site was not altered, and this correlated with an inability of the GG-NER complex to reposition nucleosomes in vitro. We discuss how the GG-NER complex might facilitate GG-NER while preventing unregulated gene transcription during this process. [ABSTRACT FROM AUTHOR]

Summary: Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility. Targeting of minicells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. This affects highly significant tumor growth inhibition and regression in mouse xenografts and case studies of lymphoma in dogs despite administration of minute amounts of drug and antibody; a factor critical for limiting systemic toxicity that should allow the use of complex regimens of combination chemotherapy. [Copyright &y& Elsevier]

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early Cl when Mcm2-7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E-deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E-mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation. [ABSTRACT FROM AUTHOR]

The article discusses the highlights of the 78th Symposium in the Cold Spring Harbor Symposia on Quantitative Biology series on the theme, "Immunity and Tolerance." The series gathers the world's scientists for a presentation of new ideas on a chosen topic in biological research. It cites topics covered in the 2013 Symposium including molecular mechanisms of B and T lymphocyte development in experimental models from the single cell to the entire organism, and from single genes to genomes.

A foreword to a 2010 issue of the journal "Cold Spring Harbor Symposia on Quantitative Biology" is presented.

Histone H3 variant H3.3, while differing from canonical H3 (H3.1) by only five amino acids, is assembled into nucleosomes, along with histone H4, at genic regions by the histone chaperone HIRA, whereas H3.1 is assembled into nucleosomes in a CAF-1-dependent reaction. Here, we show that phosphorylation of histone H4 Ser 47 (H4S47ph), catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by increasing the binding affinity of HIRA to H3.3-H4 and reducing association of CAF-1 with H3.1-H4. These results reveal a mechanism whereby H4S47ph distinctly regulates nucleosome assembly of H3.1 and H3.3. [ABSTRACT FROM AUTHOR]

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC . This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria., Competing Interests: Competing interests statement:The author declares no competing interest.

Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication. Origin specification and configuration vary substantially between eukaryotic species and in some cases co-evolved with gene-silencing mechanisms. We discuss the possibility that centromeres and origins of DNA replication were originally derived from a common element and later separated during evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Saccharomyces cerevisiae Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions., Competing Interests: YS, RK, JG No competing interests declared, BS Reviewing editor, eLife, (© 2022, Sheu et al.)

As a cell prepares to divide, a molecular actor known as the Origin Recognition Complex makes intricate ATP-driven movements to recruit proteins required to duplicate DNA., Competing Interests: BS No competing interests declared, (© 2022, Stillman.)

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization., Competing Interests: HC, KB, OD, OK, KH, SA, PA, HA, KC, CV, MS, WM No competing interests declared, BS Reviewing editor, eLife, (© 2021, Chou et al.)

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals with RMS, including all those diagnosed as adults, those diagnosed with fusion-positive tumors during childhood (including metastatic and nonmetastatic tumors), and those diagnosed with metastatic disease during childhood (including fusion-positive and fusion-negative tumors). This white paper outlines current challenges in RMS research and their implications for developing more effective therapies. Urgent clinical problems include local control, systemic disease, need for improved risk stratification, and characterization of differences in disease course in children and adults. Biological challenges include definition of the cellular functions of PAX-FOXO1 fusion proteins, clarification of disease heterogeneity, elucidation of the cellular origins of RMS, delineation of the tumor microenvironment, and identification of means for rational selection and testing of new combination therapies. To streamline future therapeutic developments, it will be critical to improve access to fresh tumor tissue for research purposes, consider alternative trial designs to optimize early clinical testing of candidate drugs, coalesce advocacy efforts to garner public and industry support, and facilitate collaborative efforts between academia and industry., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.

The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle.

The origin recognition complex (ORC) was first discovered in the baker's yeast in 1992. Identification of ORC opened up a path for subsequent molecular level investigations on how eukaryotic cells initiate and control genome duplication each cell cycle. Twenty years after the first biochemical isolation, ORC is now taking on a three-dimensional shape, although a very blurry shape at the moment, thanks to the recent electron microscopy and image reconstruction efforts. In this chapter, we outline the current biochemical knowledge about ORC from several eukaryotic systems, with emphasis on the most recent structural and biochemical studies. Despite many species-specific properties, an emerging consensus is that ORC is an ATP-dependent machine that recruits other key proteins to form pre-replicative complexes (pre-RCs) at many origins of DNA replication, enabling the subsequent initiation of DNA replication in S phase.

Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4-Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.

The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC-Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1-5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit-subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1-DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.

In eukaryotic cells the concentration of dNTP is highest in S phase and lowest in G1 phase and is controlled by ribonucleotide reductase (RNR). RNR activity is eliminated in all eukaryotes in G1 phase by a variety of mechanisms: transcriptional regulation, small inhibitory proteins, and protein degradation. After activation of RNR upon commitment to S phase, dATP feedback inhibition ensures that the dNTP concentration does not exceed a certain maximal level. It is not apparent why limitation of dNTP concentration is necessary in G1 phase. In principle, dATP feedback inhibition should be sufficient to couple dNTP production to utilization. We demonstrate that in Saccharomyces cerevisiae constitutively high dNTP concentration transiently arrests cell cycle progression in late G1 phase, affects activation of origins of replication, and inhibits the DNA damage checkpoint. We propose that fluctuation of dNTP concentration controls cell cycle progression and the initiation of DNA replication.

The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromo-adjacent homology (BAH) domain found in various chromatin-associated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-A cocrystal structure of the N-terminal domain of Orc1p in complex with the Orc1p-interacting domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting domain has a bilobal structure: an alpha/beta N-terminal lobe and a C-terminal lobe resembling the Tudor domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subdomain and the BAH domain of Orc1p. The structure provides a mechanistic understanding of Orc1p-Sir1p interaction specificity, as well as insights into protein-protein interactions involving BAH domains in general.

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1) and mini-chromosome maintenance (MCM) proteins. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation, a process referred to as 'replication licensing'. We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre-RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle. The process of DNA replication must also be carefully coordinated with other cell-cycle processes including mitosis and cytokinesis. Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell-cycle transitions. Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication.