RESUMO
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.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Reconhecimento de Origem , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.
Assuntos
Endoderma/enzimologia , Evolução Molecular , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Vertebrados/genética , Substituição de Aminoácidos , Animais , Aurora Quinase B/metabolismo , Feminino , Humanos , Camundongos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Vertebrados/metabolismoRESUMO
Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.
Assuntos
Replicação do DNA/genética , Recombinação Homóloga/genética , Mitose/genética , Estresse Oxidativo/genética , Acetilcisteína/farmacologia , Animais , Células CHO , Centrossomo/efeitos dos fármacos , Cricetulus , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes/genética , Histonas/genética , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Imagem Individual de Molécula , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Homologous recombination deficient (HR(-)) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR(-) cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR(-) cells. Reciprocally, supplying nucleotide precursors to HR(-) cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.
Assuntos
Instabilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Recombinação Homóloga/fisiologia , Mitose/fisiologia , Animais , Afidicolina , Linhagem Celular , Centrossomo/fisiologia , Segregação de Cromossomos/fisiologia , Cricetinae , Cricetulus , Citometria de Fluxo , Hidroxiureia/metabolismo , Microscopia de Vídeo , Estatísticas não Paramétricas , Imagem com Lapso de TempoRESUMO
Alterations of the dynamics of DNA replication cause genome instability. These alterations known as "replication stress" have emerged as a major source of genomic instability in pre-neoplasic lesions, contributing to cancer development. The concept of replication stress covers a wide variety of events that distort the temporal and spatial DNA replication program. These events have endogenous or exogenous origins and impact globally or locally on the dynamics of DNA replication. They may arise within a short window of time (acute stress) or during each S phase (chronic stress). Here, we review the known situations in which the dynamics of DNA replication is distorted. We have united them in four main categories: (i) inadequate firing of replication origins (deficiency or excess), (ii) obstacles to fork progression, (iii) conflicts between replication and transcription and (iv) DNA replication under inappropriate metabolic conditions (unbalanced DNA replication). Because the DNA replication program is a process tightly regulated by many factors, replication stress often appears as a cascade of events. A local stress may prevent the completion of DNA replication at a single locus and subsequently compromise chromosome segregation in mitosis and therefore have a global effect on genome integrity. Finally, we discuss how replication stress drives genome instability and to what extent it is relevant to cancer biology.
Assuntos
Neoplasias/genética , Animais , Segregação de Cromossomos , Replicação do DNA , Instabilidade Genômica , Humanos , Mitose , Neoplasias/patologia , Transcrição GênicaRESUMO
The faithful transmission of genetic information to daughter cells is central to maintaining genomic stability and relies on the accurate and complete duplication of genetic material during each cell cycle. However, the genome is routinely exposed to endogenous and exogenous stresses that can impede the progression of replication. Such replication stress can be an early cause of cancer or initiate senescence. Replication stress, which primarily occurs during S phase, results in consequences during mitosis, jeopardizing chromosome segregation and, in turn, genomic stability. The traces of replication stress can be detected in the daughter cells during G1 phase. Alterations in mitosis occur in two types: 1) local alterations that correspond to breaks, rearrangements, intertwined DNA molecules or non-separated sister chromatids that are confined to the region of the replication dysfunction; 2) genome-wide chromosome segregation resulting from centrosome amplification (although centrosomes do not contain DNA), which amplifies the local replication stress to the entire genome. Here, we discuss the endogenous causes of replication perturbations, the mechanisms of replication fork restart and the consequences for mitosis, chromosome segregation and genomic stability.