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1.
Cell ; 180(6): 1228-1244.e24, 2020 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-32142649

RESUMEN

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.


Asunto(s)
Reparación del ADN/fisiología , ARN Polimerasa II/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN/metabolismo , Daño del ADN/fisiología , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Polimerasa II/genética , Ubiquitinación
2.
Hum Mol Genet ; 32(9): 1439-1456, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-36458887

RESUMEN

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.


Asunto(s)
Histonas , Enfermedades de Inmunodeficiencia Primaria , Humanos , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN/genética , ADN/metabolismo , Metilación de ADN/genética , Metilación de ADN/fisiología , Células HEK293 , Histonas/genética , Histonas/metabolismo , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/metabolismo , Mutación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Inestabilidad Cromosómica/genética , Inestabilidad Cromosómica/fisiología , Centrómero/genética , Centrómero/metabolismo , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/metabolismo , Cara/anomalías , Genoma Humano/genética , Genoma Humano/fisiología
3.
Cancer Sci ; 115(7): 2125-2137, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38651239

RESUMEN

Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics.


Asunto(s)
Carcinogénesis , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Neoplasias , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Carcinogénesis/genética , Neoplasias/genética , Neoplasias/patología , Animales , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Ubiquitinación , Mutagénesis , Reparación del ADN/genética
4.
EMBO J ; 37(9)2018 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-29581097

RESUMEN

Polymerase-blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single-stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re-priming downstream of lesions can give rise to daughter-strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9-dependent mechanism of damage signaling is distinct from the Mrc1-dependent, fork-associated response to replication stress induced by conditions such as nucleotide depletion or replisome-inherent problems, but reminiscent of replication-independent checkpoint activation by single-stranded DNA Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase-blocking lesions mainly emanates from Exo1-processed, postreplicative daughter-strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signaling.


Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Replicación del ADN/fisiología , ADN de Hongos/biosíntesis , Fase S/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
PLoS Genet ; 13(5): e1006789, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28481910

RESUMEN

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.


Asunto(s)
Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , Schizosaccharomyces/genética , Ubiquitinación , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Schizosaccharomyces/metabolismo
6.
Nature ; 465(7300): 951-5, 2010 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-20453836

RESUMEN

Post-replication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required for an error-free pathway that probably involves a template switch to the undamaged sister chromatid. Both the timing of PRR events during the cell cycle and their location relative to replication forks, as well as the factors required downstream of PCNA ubiquitylation, have remained poorly characterized. Here we demonstrate that the RAD6 pathway normally operates during S phase. However, using an inducible system of DNA damage bypass in budding yeast (Saccharomyces cerevisiae), we show that the process is separable in time and space from genome replication, thus allowing direct visualization and quantification of productive PRR tracts. We found that both during and after S phase ultraviolet-radiation-induced lesions are bypassed predominantly via translesion synthesis, whereas the error-free pathway functions as a backup system. Our approach has revealed the distribution of PRR tracts in a synchronized cell population. It will allow an in-depth mechanistic analysis of how cells manage the processing of lesions to their genomes during and after replication.


Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Genoma/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , Ciclo Celular/fisiología , Cromatina/metabolismo , Daño del ADN/efectos de la radiación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Tiempo , Enzimas Ubiquitina-Conjugadoras , Rayos Ultravioleta
7.
Mol Cell ; 29(5): 625-36, 2008 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-18342608

RESUMEN

Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.


Asunto(s)
Daño del ADN , ADN de Cadena Simple/metabolismo , Proteína de Replicación A/metabolismo , Ubiquitina/metabolismo , Animales , Ciclo Celular/fisiología , Línea Celular , Replicación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/genética
8.
Nucleic Acids Res ; 42(19): e146, 2014 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-25106872

RESUMEN

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.


Asunto(s)
Proteínas de Unión al ADN/análisis , Microscopía Fluorescente/métodos , Ciclo Celular , Replicación del ADN , Difusión , Componente 4 del Complejo de Mantenimiento de Minicromosoma/análisis , Antígeno Nuclear de Célula en Proliferación/análisis , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análisis
9.
DNA Repair (Amst) ; 141: 103740, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39096696

RESUMEN

An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.


Asunto(s)
Replicación del ADN , Humanos , ADN/metabolismo , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , ADN Polimerasa II/metabolismo , Eucariontes/enzimología , Eucariontes/genética , ADN Polimerasa III/metabolismo , Células Eucariotas/metabolismo , Células Eucariotas/enzimología , ADN Polimerasa I/metabolismo
10.
Cell Genom ; 4(8): 100610, 2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-39053455

RESUMEN

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.


Asunto(s)
Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Humanos , Replicación del ADN , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Duplicación de Gen , Origen de Réplica/genética , Roturas del ADN de Doble Cadena , Sistemas CRISPR-Cas/genética
11.
bioRxiv ; 2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38352431

RESUMEN

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD , a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTS: Expression of NELFCD transcript is up-regulated in colorectal tumors NELF-C protein is mandatory for the transition between G1-S phases during cell cycleNELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausingNELF-C loss leads Pol II to invade DNA replication initiation zones.

12.
Phys Rev E ; 107(5-1): 054404, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37329042

RESUMEN

Self-replicability is a unique attribute observed in all living organisms, and the question of how the life was physically initiated could be equivalent to the question of how self-replicating informative polymers were formed in the abiotic material world. It has been suggested that the present DNA and proteins world was preceded by an RNA world in which genetic information of RNA molecules was replicated by the mutual catalytic function of RNA molecules. However, the important question of how the transition occurred from a material world to the very early pre-RNA world remains unsolved both experimentally and theoretically. We present an onset model of mutually catalytic self-replicative systems formed in an assembly of polynucleotides. A quantitative expression of the critical condition for the onset of growing fluctuation towards self-replication in this model is obtained by analytical and numerical calculations.


Asunto(s)
Polinucleótidos , ARN , Polinucleótidos/genética , ARN/genética , ADN/genética
13.
Sci Adv ; 9(9): eadd9742, 2023 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-36867705

RESUMEN

Secretory pathway Ca2+/Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca2+/Mn2+-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+-binding site in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+/Mn2+ transport.


Asunto(s)
Adenosina Trifosfatasas , Aparato de Golgi , Humanos , Microscopía por Crioelectrón , Sitios de Unión , Adenosina Trifosfato , ATPasas Transportadoras de Calcio
14.
Nat Commun ; 13(1): 7221, 2022 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-36434012

RESUMEN

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics.


Asunto(s)
ADN Polimerasa Dirigida por ADN , Genoma Humano , Humanos , Genoma Humano/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Replicación del ADN/genética
15.
DNA Repair (Amst) ; 82: 102688, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31450086

RESUMEN

In eukaryotes, the DNA damage bypass pathway is promoted by ubiquitylation of PCNA at the conserved lysine 164. Using CRISPR-Cas9 system, we introduced amino acid substitution at K165 of C. elegans PCNA that corresponds to K164 in other characterised organisms and examined the contribution of this residue at a variety of stages during development. In the presence of UV-induced DNA lesions, PCNA-K165 is crucial for not only the early embryonic stages but also during larval development, implicating its functions for a broad time period during animal development. We also show that, without induction of DNA damage, concomitant inhibition of PCNA ubiquitylation and checkpoint activation causes abnormal gametogenesis events and severely impairs reproduction of worms. Our findings suggest a conserved function of PCNA ubiquitylation in tolerance of UV-induced damage and also propose that PCNA ubiquitylation contributes to gametogenesis during unperturbed C. elegans development.


Asunto(s)
Sustitución de Aminoácidos , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Desarrollo Embrionario/genética , Gametogénesis/genética , Antígeno Nuclear de Célula en Proliferación/genética , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Daño del ADN , Epistasis Genética , Enzimas Ubiquitina-Conjugadoras/genética
16.
Methods Mol Biol ; 1672: 239-259, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29043629

RESUMEN

Mapping the usage of replicative DNA polymerases has previously proved to be technically challenging. By exploiting mutant polymerases that incorporate ribonucleotides into the DNA with a significantly higher proficiency than their wild-type counterparts, we and others have developed methods that can identify what proportion of each DNA strand (i.e., the Watson and Crick strands) is replicated by a specific DNA polymerase. The incorporation of excess ribonucleotides by a mutated polymerase effectively marks, in each individual cells, the DNA strand that is replicated by that specific mutated polymerase. Changes to DNA polymerase usage can be examined at specific loci by Southern blot analysis while a global analysis of polymerase usage can be achieved by applying next-generation sequencing. This genome-wide data also provides a direct measure of replication origin efficiency and can be used to indirectly calculate replication timing.


Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Ribonucleótidos , Biología Computacional/métodos , División del ADN , ADN de Hongos , Genoma Fúngico , Secuenciación de Nucleótidos de Alto Rendimiento , Origen de Réplica , Saccharomyces cerevisiae/genética , Programas Informáticos
17.
Genes Genet Syst ; 82(1): 35-42, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17396018

RESUMEN

Using the CAN1 gene in haploid cells or heterozygous diploid cells, we characterized the effects of mutations in the RAD52 and REV3 genes of Saccharomyces cerevisiae in spontaneous mutagenesis. The mutation rate was 5-fold higher in the haploid rad52 strain and 2.5-fold lower in rev3 than in the wild-type strain. The rate in the rad52 rev3 strain was as low as in the wild-type strain, indicating the rad52 mutator phenotype to be dependent on REV3. Sequencing indicated that G:C-->T:A and G:C-->C:G transversions increased in the rad52 strain and decreased in the rev3 and rad52 rev3 strains, suggesting a role for REV3 in transversion mutagenesis. In diploid rev3 cells, frequencies of can1Delta::LEU2/can1Delta::LEU2 from CAN1/can1Delta::LEU2 due to recombination were increased over the wild-type level. Overall, in the absence of RAD52, REV3-dependent base-substitutions increased, while in the absence of REV3, RAD52-dependent recombination events increased. We further found that the rad52 mutant had an increased rate of chromosome loss and the rad52 rev3 double mutant had an enhanced chromosome loss mutator phenotype. Taken together, our study indicates that the error-free RAD52 pathway and error-prone REV3 pathway for rescuing replication fork arrest determine spontaneous mutagenesis, recombination, and genome instability.


Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Mutagénesis/fisiología , Mutación , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Análisis Mutacional de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Diploidia , Haploidia , Pérdida de Heterocigocidad , Fenotipo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/genética
18.
Mutat Res ; 617(1-2): 90-7, 2007 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-17289091

RESUMEN

Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.


Asunto(s)
Aneuploidia , Compuestos de Bifenilo/metabolismo , Carcinógenos/metabolismo , Hidroquinonas/metabolismo , Saccharomyces cerevisiae/genética , Tubulina (Proteína)/metabolismo , División Celular , Cromosomas Fúngicos/genética , Citometría de Flujo , Fase G1 , Pérdida de Heterocigocidad , Microtúbulos/metabolismo
19.
Mutat Res ; 600(1-2): 177-83, 2006 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-16737721

RESUMEN

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.


Asunto(s)
Pérdida de Heterocigocidad , Fase S , Saccharomyces cerevisiae/genética , Rayos Ultravioleta , Ciclo Celular/efectos de la radiación , ADN/metabolismo , ADN/efectos de la radiación , Reparación del ADN , Modelos Genéticos , Fase S/genética , Fase S/efectos de la radiación , Saccharomyces cerevisiae/efectos de la radiación , Factores de Tiempo
20.
Nat Protoc ; 10(11): 1786-801, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26492137

RESUMEN

Ribonucleotides are frequently misincorporated into DNA during replication, and they are rapidly repaired by ribonucleotide excision repair (RER). Although ribonucleotides in template DNA perturb replicative polymerases and can be considered as DNA damage, they also serve positive biological functions, including directing the orientation of mismatch repair. Here we describe a method for ribonucleotide identification by high-throughput sequencing that allows mapping of the location of ribonucleotides across the genome. When combined with specific mutations in the replicative polymerases that incorporate ribonucleotides at elevated frequencies, our ribonucleotide identification method was adapted to map polymerase usage across the genome. Polymerase usage sequencing (Pu-seq) has been used to define, in unprecedented detail, replication dynamics in yeasts. Although other methods that examine replication dynamics provide direct measures of replication timing and indirect estimates of origin efficiency, Pu-seq directly ascertains origin efficiency. The Pu-seq protocol can be completed in 12-14 d.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/química , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ribonucleótidos/análisis , Replicación del ADN
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