The Mcm2-Ctf4-Polα Axis Facilitates Parental Histone H3-H4 Transfer to Lagging Strands

Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4) 2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Polα axis facilitates the transfer of parental (H3-H4) 2 tetramers to lagging-strand DNA at replication forks. Mutating the conserved histone-binding domain of the Mcm2 subunit of the CMG (Cdc45-MCM-GINS) DNA helicase, which translocates along the leading-strand template, results in a marked enrichment of parental (H3-H4) 2 on leading strand, due to the impairment of the transfer of parental (H3-H4) 2 to lagging strands. Similar effects are observed in Ctf4 and Polα primase mutants that disrupt the connection of the CMG helicase to Polα that resides on lagging-strand template. Our results support a model whereby parental (H3-H4) 2 complexes displaced from nucleosomes by DNA unwinding at replication forks are transferred by the CMG-Ctf4-Polα complex to lagging-strand DNA for nucleosome assembly at the original location. How parental histone H3-H4 tetramers are transferred to replicating DNA strands for epigenetic inheritance remains largely unknown. Gan et al. show that parental H3-H4 tetramers bind to Mcm2, which travels along the leading-strand template, and are then transferred to the lagging strand by the Mcm2-Ctf4-Polα complex for nucleosome assembly. </p

Chromatin assembly factor-1 preserves genome stability in ctf4∆ cells by promoting sister chromatid cohesion

Chromatin assembly and the establishment of sister chromatid cohesion are intimately connected to the progression of DNA replication forks. Here we examined the genetic interaction between the heterotrimeric chromatin assembly factor-1 (CAF-1), a central component of chromatin assembly during replication, and the core replisome component Ctf4. We find that CAF-1 deficient cells as well as cells affected in newly-synthesized H3-H4 histones deposition during DNA rep-lication exhibit a severe negative growth with ctf4∆ mutant. We dissected the role of CAF-1 in the maintenance of genome stability in ctf4∆ yeast cells. In the absence of CTF4, CAF-1 is essential for viability in cells experiencing replication problems, in cells lacking functional S-phase checkpoint or functional spindle checkpoint, and in cells lacking DNA repair pathways involving homologous recombination. We present evidence that CAF-1 affects cohesin association to chromatin in a DNA-damage-dependent manner and is essential to maintain cohesion in the absence of CTF4. We also show that Eco1-catalyzed Smc3 acetylation is reduced in absence of CAF-1. Furthermore, we describe genetic interactions between CAF-1 and essential genes involved in cohesin loading, cohesin stabilization, and cohesin component indicating that CAF-1 is crucial for viability when sister chromatid cohesion is affected. Finally, our data indicate that the CAF-1-dependent pathway required for cohesion is functionally distinct from the Rtt101-Mms1-Mms22 pathway which functions in replicated chromatin assembly. Collectively, our results suggest that the deposition by CAF-1 of newly-synthesized H3-H4 histones during DNA replication creates a chromatin environment that favors sister chromatid cohesion and maintains genome integrity

Symmetric inheritance of parental histones governs epigenome maintenance and embryonic stem cell identity

Modified parental histones are segregated symmetrically to daughter DNA strands during replication and can be inherited through mitosis. How this may sustain the epigenome and cell identity remains unknown. Here we show that transmission of histone-based information during DNA replication maintains epigenome fidelity and embryonic stem cell plasticity. Asymmetric segregation of parental histones H3-H4 in MCM2-2A mutants compromised mitotic inheritance of histone modifications and globally altered the epigenome. This included widespread spurious deposition of repressive modifications, suggesting elevated epigenetic noise. Moreover, H3K9me3 loss at repeats caused derepression and H3K27me3 redistribution across bivalent promoters correlated with misexpression of developmental genes. MCM2-2A mutation challenged dynamic transitions in cellular states across the cell cycle, enhancing naïve pluripotency and reducing lineage priming in G1. Furthermore, developmental competence was diminished, correlating with impaired exit from pluripotency. Collectively, this argues that epigenetic inheritance of histone modifications maintains a correctly balanced and dynamic chromatin landscape able to support mammalian cell differentiation

Role of Uhrf1 and histone H3 ubiquitination in chromatin re-establishment during DNA replication

Chromatin re-establishment after DNA replication consists of nucleosome re-assembly and restoration of epigenetics marks in both daughter strands. These marks comprise DNA methylation and post-translational modifications of histones that regulate chromatin conformation and gene expression. Unrepaired errors during these processes result in genomic instability that leads to diseases such as cancer. The E3 ubiquitin ligase Uhrf1 is an epigenetic regulator considered an oncogene for its ability to promote cell proliferation. This protein mediates DNA methylation maintenance by ubiquitinating histone H3 and Paf15 and subsequently recruiting DNA methyl transferase Dnmt1 to hemi-methylated sites. Although the role of Uhrf1 in recruiting Dnmt1 is clear, there are still many unresolved questions about the mechanism itself and how it is coordinated with other processes taking place at the same time, such as chromatin assembly and remodelling. This PhD project focuses on understanding the roles of Uhrf1 and histone H3 ubiquitination in DNA methylation maintenance and chromatin re-establishment during DNA replication. Using Xenopus laevis egg extract as a model system, I have confirmed the role of Uhrf1 in mediating the dual mono-ubiquitination of histone H3 and Paf15 and recruiting Dnmt1 through ubiquitination. Importantly, this study has revealed that Uhrf1 also recruits Lig1 to replicating chromatin and its absence impairs the binding of Fen1 and CAF1. Finally, I have identified novel interacting partners of Uhrf1 during DNA replication. All these findings suggest that Uhrf1 plays a central and multifaceted role in chromatin-re-establishment, coordinating DNA methylation with DNA replication and nucleosome assembly

Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage.

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template

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

Investigating DNA Polymerase Alpha in the C. elegans germline

Understanding the mechanisms behind adult stem cell maintenance and division is an important effort in medicine for developing treatments for a multitude of illnesses. Asymmetric stem cell division offers many insights on how stem cells can maintain their potency while still providing cellular division to aid in regeneration and repair of damaged tissue. Applying asymmetric stem cell mechanisms to symmetrically diving systems may offer a way to genetically induce maintained potency when there normally wouldn’t be. In this study, our goal is to induce stemness—a phenomenon observed in Drosophila melanogaster via heterozygous mutations in lagging-strand DNA polymerase α (Polα) or through a small-molecule Polα inhibitor—in the symmetrically dividing germline stem cell population of Caenorhabditis elegans. We accomplish this by introducing a heterozygous deletion for DNA Polymerase Alpha (POLA-1) in order to create delayed lagging strand synthesis during stem cell replication. We find that genetically reducing the levels of POLA-1 via the heterozygous deletion result in sustained germline stem cell maintenance in physiological conditions such as aging and pathological conditions such as an increased resistance to acute pathogen infection. Furthermore, we demonstrate that significant effects of reducing POLA-1 are dependent on the homeostasis of the worm

Understanding the function of the Rtt101 E3 ubiquitin ligase in response to replication stress

The duplication of the cellular genetic material has to be precisely regulated to maintain genome integrity. Damage caused by a wide range of exogenous factors, commonly summarized as replication stress, interferes with DNA replication. Genome integrity is further threatened by endogenously arising structures such as ribonucleotide monophosphates (rNMP) that are frequently misincorporated into genomic DNA. They are removed by the RNase H2 enzyme in a process termed ribonucleotide excision repair (RER). When RER is defective, rNMPs accumulate in the genome and induce replication stress. Cullin 4 (CUL4)-based E3 ubiquitin ligases are required for DNA replication and repair in the presence of replicative DNA damage. In Saccharomyces cerevisiae, the CUL4 ortholog Rtt101 promotes DNA replication through damaged templates. However, the underlying mechanism and relevant ubiquitylation targets are poorly understood. In this thesis we characterized the mechanism by which Rtt101 promotes DNA replication in the presence of the alkylating drug methyl methanesulfonate (MMS). We found that Rtt101, in complex with the putative substrate adaptor Mms22 (Rtt101-Mms22), counteracts a replicative function of the replisome component Mrc1. However, this does not alter Mrc1 protein levels. Instead, our genetic data suggests that interactions of Mrc1 with other replisome proteins are modulated. We propose that Rtt101 allows recombination-mediated fork restart at MMS-induced DNA lesions. We further uncovered a novel role of Rtt101-Mms22 in the tolerance of misincorporated rNMPs that accumulate in the absence of RER. Cells lacking both Rtt101 and RER display reduced viability, which is not caused by increased levels of genomic rNMPs and can be partially offset by deletion of MRC1. Ubiquitin remnant profiling, a mass spectrometry-based approach, identified the leading strand polymerase ε subunit Dpb2 as a potential target of Rtt101. We suggest that Rtt101 ubiquitylates Dpb2 at replication forks that stall or break due to unrepaired rNMPs. This Rtt101-dependent ubiquitylation might facilitate replication fork restart or DNA synthesis repriming downstream of the site of damage. We present evidence that underlines an important function of the Rtt101 E3 ubiquitin ligase to tolerate several aspects of faulty RER. Our data indicates a similar mechanism as under genotoxin-induced replication stress.Die Duplizierung des zellulären genetischen Materials muss genau reguliert werden, um die Integrität des Genoms aufrechtzuerhalten. Schäden, die durch eine Vielzahl von exogenen Faktoren verursacht und üblicherweise als Replikationsstress zusammengefasst werden, können die DNA-Replikation beeinträchtigen. Die Integrität des Genoms wird weiterhin durch endogen auftretende Strukturen wie Ribonukleotidmonophosphate (rNMP) bedroht, die häufig in genomische DNA eingebaut werden. Sie werden durch das Enzym RNase H2 in einem als Ribonukleotid-Exzisionsreparatur (RER) bezeichneten Prozess entfernt. Wenn RER defekt ist, akkumulieren rNMPs im Genom und induzieren Replikationsstress. Cullin 4 (CUL4) -basierte E3-Ubiquitin-Ligasen sind für die DNA-Replikation und -Reparatur in Gegenwart von replikativen DNA-Schäden erforderlich. In Saccharomyces cerevisiae fördert das CUL4-Ortholog Rtt101 die DNA-Replikation durch beschädigte DNA Matrizen. Der zugrundeliegende Mechanismus und die relevanten Ziele der Ubiquitylierung sind jedoch kaum erforscht. In dieser Dissertation haben wir den Mechanismus charakterisiert, durch den Rtt101 die DNA-Replikation in Gegenwart des alkylierenden Chemotherapeutikums Methylmethansulfonat (MMS) fördert. Wir haben herausgefunden, dass Rtt101 im Komplex mit dem mutmaßlichen Substratadapter Mms22 (Rtt101-Mms22) einer replikativen Funktion des Replisombestandteils Mrc1 entgegenwirkt. Dies ändert jedoch nicht die Mengen an Mrc1-Protein. Stattdessen legen unsere genetischen Daten nahe, dass Wechselwirkungen zwischen Mrc1 und anderen Replisomproteinen moduliert werden. Wir schlagen vor, dass Rtt101 einen Rekombinations-vermittelten Neustart der Replikationsgabel an MMS-induzierten DNA-Läsionen ermöglicht. Wir haben außerdem eine neue Rolle von Rtt101-Mms22 in der Toleranz von fälschlicherweise eingebauten rNMPs aufgedeckt, die sich in Abwesenheit von RER anhäufen. Zellen, in welchen sowohl Rtt101 als auch RER fehlt, zeigen eine verminderte Lebensfähigkeit, die nicht durch erhöhte Mengen an genomischen rNMPs verursacht wird und durch die Deletion von MRC1 teilweise ausgeglichen werden kann. Mithilfe der Profilerstellung von Ubiquitinresten, einem auf Massenspektrometrie basierenden Ansatz, haben wir Dpb2, eine Untereinheit von DNA-Polymerase ε am kontinuierlich replizierten Strang, als potentielles Ziel von Rtt101 identifiziert. Wir schlagen vor, dass Rtt101 Dpb2 an Replikationsgabeln ubiquityliert, die aufgrund von nicht reparierten rNMPs ins Stocken geraten oder gebrochen sind. Diese Rtt101-abhängige Ubiquitylierung könnte den Neustart der Replikationsgabel oder die Wiederaufnahme der DNA-Synthese hinter der Schadensstelle erleichtern. Wir präsentieren Beweise, die eine wichtige Funktion der Rtt101 E3-Ubiquitin-Ligase unterstreichen, die benötigt wird um verschiedene Aspekte fehlerhafter RER zu tolerieren. Unsere Daten weisen auf einen ähnlichen Mechanismus wie unter Genotoxin-induziertem Replikationsstress hin.VI, 169 Seite

Mass spectrometric approaches to study the composition and assembly of centromere associated complexes

Mitosis is the process of dividing a eukaryotic cell into two identical daughter cells. This part of the cell cycle executes the faithful propagation of the genome. A prerequisite for maintaining genome stability is the assembly of the conserved kinetochore structure at chromosomal loci called centromeres. The kinetochore is a macromolecular protein complex that physically links chromosomes to spindle microtubules. Aberrations in chromosome segregation cause aneuploidy, which has been associated with tumorigenesis, trisomy, and age-related pathologies. To ensure the accurate segregation of sister chromatids, their kinetochores have to be attached to microtubules emanating from opposite spindle poles, a configuration which is known as biorientation of chromosomes. The kinetochore is composed of more than 80 proteins, which are organized in stable subcomplexes and follow a conserved hierarchy of assembly from centromeric chromatin to microtubules: the centromere proximal inner kinetochore or Constitutive Centromere Associated Network (CCAN), the microtubule binding KMN (KNL1/MIS12/NDC80) network at the outer kinetochore and the fibrous corona. The proteins of the CCAN complex build the interface between centromeric chromatin and the microtubule-binding unit. Several kinetochore proteins are conserved among eukaryotes. In contrast, the underlying centromeric chromatin is highly divergent and epigenetically specified. The major epigenetic mark of the centromere are nucleosomes that have H3 replaced by centromere specific histone variant CENP A. Interestingly, the levels of CENP-A are halved during DNA replication by equally distributing CENP-A between sister chromatids. Cells pass through mitosis with half-maximal CENP-A levels until they are replenished during mitotic exit. The underlying molecular pathways of histone redistribution during DNA replication and CENP-A replenishment in the early G1-phase remain largely unknown. In this thesis, I analyzed the protein composition of the human centromere in a time-resolved manner to study the quantitative changes in protein interactions of CENP-A containing oligo-nucleosomes. This proteomic screen detected several proteins that are associated with the centromere in a cell cycle-dependent manner and identified candidates that may regulate CENP-A distribution to the leading and lagging DNA strands subsequent to replication. Besides chromatin-associated proteins, histone remodelers, and readers and writers of histone post-translational modifications (PTMs), I identified an uncharacterized protein. This transcription factor-like protein was selectively associated with CENP-A at levels comparable to CCAN proteins throughout the entire cell cycle, indicating that this protein may have a structural role at the centromere or inner kinetochore. Spatial restraints derived from the mass spectrometric analysis of crosslinked proteins (XLMS) are widely applied in integrative structural biology approaches to determine protein connectivity. I used label-free quantification of crosslink spectral data to show the dependence of crosslink distances and intensities, which facilitated the estimation of protein dissociation constants and aided the prediction of interfaces of budding yeast subunit contacts. The load-bearing link of chromosomes to microtubules through the kinetochore is stabilized through phosphorylation of CCAN and KMN proteins by mitotic kinases. Titration of the assembly of up to 11 budding yeast kinetochore proteins in vitro indicated that phosphorylation of CCAN and KMN proteins induces cooperative stabilization of the kinetochore at the centromeric nucleosome, which is required to withstand the pulling forces of depolymerizing microtubules. Phosphorylation of distinct sites at the outer kinetochore subunit Dsn1 by AuroraBIpl1, and at the inner kinetochore protein Mif2, mediated cooperativity of the kinetochore assembly. These phosphorylation events decreased the KD values of the kinetochore protein-interactions to the centromeric nucleosome by ~200-fold, which was essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modeling