The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication

The GINS complex, originally discovered in Saccharomyces cerevisiae and Xenopus laevis, binds to DNA replication origins shortly before the onset of S phase and travels with the replication forks after initiation. In this study we present a detailed characterization of the human GINS (hGINS) homolog. Using new antibodies that allow the detection of endogenous hGINS in cells and tissues, we have examined its expression, abundance, subcellular localization and association with other DNA replication proteins. Expression of hGINS is restricted to actively proliferating cells. During the S phase, hGINS becomes part of a Cdc45–MCM–GINS (CMG) complex that is assembled on chromatin. Down-regulation of hGINS destabilizes CMG, causes a G1–S arrest and slows down ongoing DNA replication, effectively blocking cell proliferation. Our data support the notion that hGINS is an essential component of the human replisome

DNA replikatsiooni initsiatsiooni uurimine Saccharomyces cerevisiaes

Väitekirja elektrooniline versioon ei sisalda publikatsioone.DNA kui eluslooduse keskne molekul kannab endas informatsiooni mis on vajalik organismi ülesehitamiseks ja funktsioneerimiseks. Selleks, et DNA-s olevast informatsioonist oleks võimalik luua elusat olendit, olgu see siis bakter või inimene, on esmalt vajalik seda informatsiooni lugeda ja edasi toimetada. Molekulaarses mõttes tähendab see seda, et DNA molekuli peal toimub pidev sagimine. Informatsiooni lugevad molekulaarsed masinad liiguvad üksteise järel mööda kromosoome ja kirjutavad selles sisalduva informatsiooni uude molekuli - RNA-sse. Saadud RNA molekulide alusel aga sünteesitakse elusate organismide peamised ehituskivid ehk valgud. Valkude ehitamiseks mõeldud informatsiooni lugemise taustal on aga vajalik tagada ka rakkude jagunemine. Selleks tuleb DNA-s sisalduv informatsioon edasi kanda kõikidesse tütarrakkudesse ja enne rakkude jagunemist olemasolevatest DNA molekulidest sünteesima uue koopia. Taaskord tuleb esmalt lugeda vanas DNA molekulis olev informatsioon ja selle alusel sünteesida uus. Siinkohal tuleb aga meeles pidada, et sünteesi aluseks oleval DNA-l on juba suur hulk teisi molekule, mis aitavad organismil funktsioneerida. DNA paljundamise masinavärk peab aga sellises olukorras, kus DNA juba on pidevas kasutuses, suutma ülima täpsusega sünteesida uue identse molekuli. Ühe eesmärgina antud uurimustöö raames uuritigi protsesse mis võimaldavad DNA täpse paljundamise sellises situatsioonis. Leidsime, et DNA kopeerimise algatamine on RNA-de pideva sünteesimise tõttu tõsiselt häiritud ja selle tõttu peab seda protsessi igas rakus korduvalt uuesti alustama. Õnneks ei kaota DNA kopeerimise masinad, mis on ajutiselt DNA molekulilt eemaldatud oma võimet uut DNA-d sünteesida. Lisaks DNA kasutamise suurele intensiivsusele raskendab DNA kasutamist ka selle molekuli suurus. Näiteks inimese DNA kogupikkus on 3 meetrit, see tuleb aga mahutada 100 mikromeetri pikkustesse rakkudesse. Selle võimaldamiseks on DNA tihedalt kokku pakitud, analoogselt niidile mis on keeratud ümber niidirulli. DNA kopeerimise algatamiseks tuleb seega esmalt leida piirkonnad mille kokkupakkimise aste on väiksem ja millele on võimalik hõlpsalt juurde pääseda. Seda protsessi uurides leidsime, et rakkudes hoitakse DNA kopeerimise algatamiseks mõeldud alad aktiivselt ligipääsetavatena. Kui neid piirkondi liigutada teistesse kromosoomidesse, jäävad nad endiselt avatuks. Sellise olukorra tagamiseks seonduvad DNA-le spetsiifilised valgud mis takistavad selle kokku pakkimist. Kui need valgud eemaldada või muteerida DNA piirkondi kuhu nad seonduvad, on DNA kopeerimise algatamine häiritud.DNA carries the information needed for building and functioning of an organism. In order to achieve this, information embeded in DNA has to be first read and edited. Molecularly, it means that multiple molecular machines are moving along chromosomes and transcribing the information into an other molecule – RNA. Based on RNA in turn, the basic building blocks of life, proteins, are being synthesized. In addition to building proteins, it is also vital for an organism to be able to divide its cells. In order to to that, the DNA content of a cell has to be duplicated. Once again, the molecular machines have to move along the chromosomes, first read and then write the information into a new DNA molecule. But remember, there is already a lot of traffic on DNA – the molecules that help to transcribe the information for building proteins. Therefore, the DNA replication machinery has to be able to cope with these obstructions and accurately copy the DNA sequence. One of the goals in this study was to determine how DNA replication manages to cope in this situation. We found that, indeed, the DNA replication initiation is disrupted from constant synthesis of the RNA molecules. On the other hand, the DNA replication machinery does not lose its functionality after being temporarily removed from DNA, and can reform shortly after. The reformed molecules can then successfully finish the job. In addition to the intensity of DNA usage, also the length of the DNA molecule makes it more difficult to copy it. For example, the length of human DNA is about 3 meters and it has to be packed into 100 micrometer long cells. In order to achieve this, DNA has to be heavily packed. Therefore, in cells the string of DNA is wound around little barrels of proteins to minimize its size. This in turn makes parts of DNA inaccessible and consequently the DNA replication machinery can not efficiently start the synthesis. When studying this phenomenon, we found that certain regions of chromosomes are being kept in a constantly unpacked state in order to efficiently start DNA synthesis. These regions are being kept in an unpacked state even when artificially moved to different chromosomes. This process is dependent on specific proteins, that bind DNA and manage to inhibit its packing. When these proteins are removed, or the DNA regions where they bind are mutated, the start of DNA replication is heavily disturbed

DNA damage responses in the context of the cell division cycle

Pagination different from approved bound copy, but content the same.During my PhD, I have investigated aspects of the DNA damage response (DDR) in the context of three different cellular scenarios: DNA damage signalling in response to double-strand breaks during mitosis, coordination of DNA replication with DNA damage responses by regulation of the GINS complex, and checkpoint activation by the prototypical checkpoint protein Rad9. Here, I show that mitotic cells treated with DNA break-inducing agents activate a ‘primary’ DDR, including ATM and DNA-PK-dependent H2AX phosphorylation and recruitment of MDC1 and the MRN complex to damage sites. However, downstream DDR events and induction of a DNA damage checkpoint are inhibited in mitosis, with full DDR activation only ensuing when damaged mitotic cells enter G1. In addition, I provide evidence that induction of a primary DDR in mitosis is biologically important for cell viability. The GINS complex is an evolutionarily conserved component of the DNA replication machinery and may represent an ideal candidate for transferring the DNA damage signal to the replication apparatus. Here, I show the identification of a consensus ‘SQ’ PIKK phosphorylation motif at the carboxyl end of the GINS complex subunit, Psf1. In Saccharomyces cerevisiae, switching the conserved serine to a glutamic acid is lethal, indicating that the site is crucial for the protein’s function. Moreover, in human cells, I identified UV-DDB, a heterodimeric complex involved in NER repair, as a binding partner that specifically interacts with the Psf1 C-terminus in vitro. Finally, I discuss my findings in characterizing functional interactions between Rad9 and Chk1 in S. cerevisiae. I show that specific consensus CDK sites within Rad9 N-terminus are essential to enable Chk1 phosphorylation and activation, and that MCPH1, a human homologue of Rad9, may share a conserved function in binding and activating Chk1, underscoring the evolutionarily conservation of checkpoint activation mechanisms

The Mechanisms of DNA Replication

DNA replication is a fundamental part of the life cycle of all organisms. Not surprisingly many aspects of this process display profound conservation across organisms in all domains of life. The chapters in this volume outline and review the current state of knowledge on several key aspects of the DNA replication process. This is a critical process in both normal growth and development and in relation to a broad variety of pathological conditions including cancer. The reader will be provided with new insights into the initiation, regulation, and progression of DNA replication as well as a collection of thought provoking questions and summaries to direct future investigations

The Mcm2-7 Replicative Helicase is Essential to Coordinate DNA replication, Checkpoint Regulation and Sister Chromatid Cohesion

DNA replication is a complex and highly regulated cellular process that ensures faithful duplication of the entire genome. To prevent genomic instability, several additional processes are coordinated with DNA replication. Eukaryotic cells employ a conserved surveillance mechanism called the S-phase checkpoint to activate a phosphorylation cascade while encountering DNA damage during DNA replication. In addition, DNA replication must also coordinate with sister chromatid cohesion, so that sister DNAs emerged from the forks are physically connected until chromosomal segregation takes place. Mcm2-7, the eukaryotic replicative helicase that unwinds dsDNA and positions at the vanguard of the replication fork, is likely the commonality among these cellular processes. In my thesis work, I find that ATP hydrolysis in one specific active site (Mcm6/2) is required to mediate DNA replication checkpoint response, sister chromatid cohesion and DNA replication initiation. Further examination reveals that a subcircuit of the checkpoint pathway including MEC1 and MRC1 and ends with Mcm2-7 is required to mediate sister chromatid cohesion. Finally, misregulation of these processes causes genomic instability and likely missegregation of chromosomes. My findings lead to a model that the regulation of ATP hydrolysis at the Mcm6/2 active site by Mrc1 modulates Mcm2/5 gate open and gate closure during initiation, DNA damage and sister chromatid cohesion

Regulatory mechanisms of mammalian replication origins

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-12-2017Esta tesis tiene embargado el acceso al texto completo hasta el 14-06-2019In mammalian cells, DNA replication starts from thousands of replication origins whose activation is tightly regulated in the cell division cycle. In this study, we have aimed at better understanding the regulation of origin selection and activation in mouse embryonic stem cells and human cancer cells. In the first part of this dissertation, we set out to investigate the dynamics of origin activation in response to stress conditions that slow down or stall replication forks. This situation promotes the activation of otherwise ‘dormant’ origins, which provide a backup mechanism to complete replication and prevent genomic instability. Using the SNS-Seq technique based on deep-sequencing of short nascent DNA strands, we have mapped the genomic positions of origins in control growth conditions and two experimental settings that trigger the activation of extra origins: (a) exposure to DNA polymerase inhibitor aphidicolin; (b) overexpression of CDC6, a limiting factor for origin licensing and activation in primary murine cells. Using SNS-Seq data we also determined the efficiency of activation of each individual origin in the cell population. Constitutive origins, in contrast to stress-responsive ones, show a strong preference towards open, transcriptionally active chromatin and display higher efficiency. Our results strongly suggest that the main response to stress is mediated by modulating the activity of pre-existing origins rather than the activation of new ones. We have also carried out an unprecedented integration of linear origin maps into 3D chromatin networks that reveals how origins tend to group together in clusters that likely correspond to DNA replication factories. Origin connections are found within the same topologically associated domain (TAD), but also between origins located in different TADs. We report for the first time that the connectivity of an origin is directly proportional to its efficiency of activation. In the second part, we aimed at dissecting a novel mechanism that regulates CDC6 protein stability. Downregulation of CDC7 kinase, known to activate the MCM helicase at replication origins, caused a drop in cellular CDC6 levels. CDC6 was phosphorylated by CDC7 in vitro and became destabilized in vivo when all possible CDC7-dependent phosphorylation sites were mutated. We report a previously unknown role of CDC7 in the regulation of CDC6 stability that is mediated by a combination of direct and indirect mechanisms.This Thesis was supported by a “La Caixa”/CNIO Fellowship from the International PhD Programme and research grants from the Spanish Ministerio de Economía y Competitividad (BFU2013-49153-P and BFU2016-80402-R

DNA replication origins in Haloferax volcanii

DNA replication is fundamental to the proliferation of life. Sites of DNA replication initiation are called replication origins. Bacteria replicate from a single origin whereas eukaryotes utilise multiple origins for each chromosome. The archaeal domain includes species which replicate using multiple origins of replication in addition to those which use a single origin. Archaeal DNA replication proteins are similar to eukaryotic replication machinery. Most characterised archaeal origins are adjacent to an orc gene which encodes a homologue of the Orc1 subunit of the eukaryotic initiator protein complex. Replication origins of the halophilic archaeon Haloferax volcanii were identified using a combination of genetic, biochemical and bioinformatic approaches. H. volcanii has a multireplicon genome consisting of a circular main chromosome and three mini-chromosomes: pHV1, pHV3 and pHV4. The major chromosome contains multiple origins of replication and is the first example of multiple origins on a single replicon in the Euryarchaeota. Each characterised origin is adjacent to an orc gene and contains repeated sequence motifs surrounding an A/T-rich duplex unwinding element. The archaeal recombinase, RadA, is homologous to eukaryotic and bacterial Rad51/RecA. It is widely held that deletion of radA results in elimination of homologous recombination. In this study the discovery of a radA-independent recombination pathway specific to replication origins is described. This dynamic mechanism was identified by observing chromosomal integration of plasmids containing H. volcanii replication origins in a radA deletion strain. The eukaryotic RAD25 gene is involved in nucleotide excision repair and transcription. H. volcanii has four RAD25 homologues, one on pHV4 and three near the oriC-2 locus on the main chromosome. A role for the assistance of oriC-2 firing is proposed based on autonomously replicating plasmid assays. Deletion of all four RAD25 homologues did not increase DNA damage sensitivity but resulted in a minor growth defect

Sld5, A Subunit of the Heterotetrameric GINS Complex is Necessary for Normal Cell Cycle Progression and Genomic Stability

Sld5 is one component of the GINS heterotetrameric complex essential to DNA replication. Specifically, GINS is known for its integral role during the G1 to S phase transition in the cell cycle. The GINS complex is comprised of multiple subunits: Psf1, Psf2, Psf3 and Sld5, all of which are highly conserved in eukaryotes. During the initiation of S phase, GINS mediates the association of multiple proteins at replication origins. SLD5 plays a central role in the GINS complex through contact with both Psf1 and Psf2. Due to this pivotal role, Sld5 is the focus of our continuing investigation into the mechanisms of DNA replication and heterochromatin formation in Drosophila. Understanding Sld5 function has employed the use of several approaches. To recognize the range of protein interactions in which SLD5 participates we are using yeast two-hybrid analysis, confirming suspected interactions. In addition to interaction studies we are utilizing two recently identified mutant alleles of SLD5 to understand its function in vivo. These p-element insertion alleles result in the truncation of the Sld5 protein removing a large portion of the C-terminal beta domain in both mutants, a domain that is believed to play a role in facilitating interactions with other proteins. The arrest point determination of Sld5 was completed and shown to occur at the late embryo/early larval stage transition of the developing Drosophila. These homozygous lethal alleles of SLD5 are being used to understand the role of Sld5 in DNA replication through EdU incorporation assays. In addition, possible roles for Sld5 in chromosome biology are being examined. These methods include the analysis of the morphology of chromosomes in polytene tissues, larval brain tissues, and embryos. Roles of Sld5 within the cell cycle have been explored by quantitation of mitotic indexes using larval brain squashes with both alleles of Sld5 showing a marked increase in mitotic figures observed when compared to wild type. In addition, Embryo analysis has revealed severe mitotic defects including asynchrony, cell dropout, and anaphase bridges are presence upon division. Exploration of the Sld5 subunit will further the understanding of the GINS complex and its role in DNA replication, along with its possible roles in chromosome biology and its role in genome maintenance.  M.S

Understanding the genomic relationship between nuclear DNA replication and genome plasticity in kinetoplastid genomes

DNA replication is an essential process in all eukaryotes initiated from sites termed origins of replication. Recent studies in the kinetoplastid species Leishmania and Trypanosoma brucei have revealed striking differences in the process of DNA replication between the largely syntenic genomes. T. brucei replication origins are generally consistent with previous eukaryotic models while Leishmania chromosomes appears to contain a single major origin, as is observed in bacteria, although how the parasites can complete replication in this manner remains unknown. Sites of DNA replication co-localise to strand switch regions where transcription initiation and termination also occur. However, not every strand switch region contains an origin of replication and differences between those containing an origin and those without have not been identified. The use of a variety of computational approaches, including machine learning, allow the investigation of origins of replication in both Leishmania and Trypanosoma brucei at the DNA sequence level and within the structure of the surrounding genomic context and further characterization of the different classes of strand switch region. A significant feature of the Leishmania genome is its ability to adapt in response to environmental pressures through copy number variation of genes and chromosomes and the formation of episomes, allowing the parasites to evade the host immune system and rapidly develop drug resistance through modulation of gene expression. Analysis of the sequence and structure of the Leishmania mexicana genome in serial passage conditions provides insight into the mechanisms underlying genome plasticity and presents a novel hypothesis explaining the potential relationship with DNA replication