Spatial regulation of DNA damage tolerance protein Rad5 interconnects genome stability maintenance and proteostasis networks.

The Rad5/HLTF protein has a central role in the tolerance to DNA damage by mediating an error-free mode of bypassing unrepaired DNA lesions, and is therefore critical for the maintenance of genome stability. We show in this work that, following cellular stress, Rad5 is regulated by relocalization into two types of nuclear foci that coexist within the same cell, which we termed 'S' and 'I'. Rad5 S-foci form in response to genotoxic stress and are associated with Rad5's function in maintaining genome stability, whereas I-foci form in the presence of proteotoxic stress and are related to Rad5's own proteostasis. Rad5 accumulates into S-foci at DNA damage tolerance sites by liquid-liquid phase separation, while I-foci constitute sites of chaperone-mediated sequestration of Rad5 at the intranuclear quality control compartment (INQ). Relocalization of Rad5 into each type of foci involves different pathways and recruitment mechanisms, but in both cases is driven by the evolutionarily conserved E2 ubiquitin-conjugating enzyme Rad6. This coordinated differential relocalization of Rad5 interconnects DNA damage response and proteostasis networks, highlighting the importance of studying these homeostasis mechanisms in tandem. Spatial regulation of Rad5 under cellular stress conditions thus provides a useful biological model to study cellular homeostasis as a whole.

Fluorescence Microscopy for Analysis of Relocalization of Structure-Specific Endonucleases.

The analysis of protein relocalization by fluorescence microscopy has been important for studying processes involved in genome integrity maintenance at the cellular level. Structure-specific endonucleases are required for genome stability, and work in budding yeast has revealed that these proteins accumulate and colocalize at discrete subnuclear foci following DNA damage. Here we describe protocols for fluorescence microscopy analysis of live budding-yeast cells containing fluorescent-tagged proteins that have been useful for the study of endonuclease relocalization during the cell cycle and under DNA-damaging conditions, all of which can be extended to the analysis of other proteins.

Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity.

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

Prevention of unwanted recombination at damaged replication forks.

Homologous recombination is essential for the maintenance of genome integrity but must be strictly controlled to avoid dangerous outcomes that produce the opposite effect, genomic instability. During unperturbed chromosome replication, recombination is globally inhibited at ongoing DNA replication forks, which helps to prevent deleterious genomic rearrangements. This inhibition is carried out by Srs2, a helicase that binds to SUMOylated PCNA and has an anti-recombinogenic function at replication forks. However, at damaged stalled forks, Srs2 is counteracted and DNA lesion bypass can be achieved by recombination-mediated template switching. In budding yeast, template switching is dependent on Rad5. In the absence of this protein, replication forks stall in the presence of DNA lesions and cells die. Recently, we showed that in cells lacking Rad5 that are exposed to DNA damage or replicative stress, elimination of the conserved Mgs1/WRNIP1 ATPase allows an alternative mode of DNA damage bypass that is driven by recombination and facilitates completion of chromosome replication and cell viability. We have proposed that Mgs1 is important to prevent a potentially harmful salvage pathway of recombination at damaged stalled forks. In this review, we summarize our current understanding of how unwanted recombination is prevented at damaged stalled replication forks.

The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks.

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.

Subnuclear Relocalization of Structure-Specific Endonucleases in Response to DNA Damage.

Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with it.

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

Rad5 plays a major role in the cellular response to DNA damage during chromosome replication.

The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5(HLTF/SHPRH), which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression of replication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.

Tolerating DNA damage during eukaryotic chromosome replication.

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage.

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.

Cell cycle-dependent regulation of the nuclease activity of Mus81-Eme1/Mms4.

The conserved heterodimeric endonuclease Mus81-Eme1/Mms4 plays an important role in the maintenance of genomic integrity in eukaryotic cells. Here, we show that budding yeast Mus81-Mms4 is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this protein occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the function of the Mus81-Mms4 complex. Consistently, a phosphorylation-defective mms4 mutant shows highly reduced nuclease activity and increases the sensitivity of cells lacking the RecQ-helicase Sgs1 to various agents that cause DNA damage or replicative stress. The mode of regulation of Mus81-Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81-Mms4 activity provides a safeguard mechanism to resolve DNA intermediates that may remain after replication and require processing before mitosis.

The S-phase checkpoint: targeting the replication fork.

The S-phase checkpoint is a surveillance mechanism, mediated by the protein kinases Mec1 and Rad53 in the budding yeast Saccharomyces cerevisiae (ATR and Chk2 in human cells, respectively) that responds to DNA damage and replication perturbations by co-ordinating a global cellular response necessary to maintain genome integrity. A key aspect of this response is the stabilization of DNA replication forks, which is critical for cell survival. A defective checkpoint causes irreversible replication-fork collapse and leads to genomic instability, a hallmark of cancer cells. Although the precise mechanisms by which Mec1/Rad53 maintain functional replication forks are currently unclear, our knowledge about this checkpoint function has significantly increased during the last years. Focusing mainly on the advances obtained in S. cerevisiae, the present review will summarize our understanding of how the S-phase checkpoint preserves the integrity of DNA replication forks and discuss the most recent findings on this topic.

Density transfer as a method to analyze the progression of DNA replication forks.

The density transfer technique is a valuable tool to examine the progression of individual DNA replication forks. It is based on the transfer of cells from a medium containing dense isotopes to a medium with light (normal) isotopes (or vice versa), to obtain DNA sequences hybrid in density that can be identified as replicated molecules. Using specific DNA probes along a chromosome, the dense isotope transfer method allows determining the extent of replication at any position of a replicon and the rate of replication fork progression. In the eukaryotic model budding yeast, this technique has been useful to establish a role for different proteins during the elongation of chromosomal replication and to analyze the movement and stability of DNA replication forks under different experimental conditions.

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA.

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.

Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1Delta with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1Delta mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1Delta mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1Delta cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae.

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

A central role for DNA replication forks in checkpoint activation and response.

The checkpoint proteins Rad53 and Mec1-Ddc2 regulate many aspects of cell metabolism in response to DNA damage. We have examined the relative importance of downstream checkpoint effectors on cell viability. Checkpoint regulation of mitosis, gene expression, and late origin firing make only modest contributions to viability. By contrast, the checkpoint is essential for preventing irreversible breakdown of stalled replication forks. Moreover, recruitment of Ddc2 to nuclear foci and subsequent activation of the Rad53 kinase only occur during S phase and require the assembly of replication forks. Thus, DNA replication forks are both activators and primary effectors of the checkpoint pathway in S phase.

Prótesis parcial removible

Principios y objetivos del tratamiento con prótesis parciales removibles. Partes de una prótesis parcial removible y sus funciones. El descanso de la prótesis parcial. Consideraciones sobre la unión del diente y el tejido. Conectores mayores. Diseño para los conectores de la base protética. Retenedores: diseño y posición. Tipos de prótesis parciales. Principios en el diseño de una prótesis parcial y la secuencia del diseño. La posición terapéutica más ventajosa. Diagnóstico y planeación del tratamiento. Preparación intrabucal. Impresiones. Comunicaciones e instrucciones con el laboratorio. Ajueste fisiológicos del armazón y los procedimientos de impresión en la técnica del modelo alterado. Relaciones y registros maxilomandibulares. Desarrollo oclusal y estético. Principios y procedimientos de inserción. Preparación del paciente e instrucciones y cuidados posteriores. Procedimientos clínicos y de laboratorio. Métodos de laboratorio. Procedimientos clínicos

Prótesis parcial removible

Principios y objetivos del tratamiento con prótesis parciales removibles. Partes de una prótesis parcial removible y sus funciones. El descanso de la prótesis parcial. Consideraciones sobre la unión del diente y el tejido. Conectores mayores. Diseño para los conectores de la base protética. Retenedores: diseño y posición. Tipos de prótesis parciales. Principios en el diseño de una prótesis parcial y la secuencia del diseño. La posición terapéutica más ventajosa. Diagnóstico y planeación del tratamiento. Preparación intrabucal. Impresiones. Comunicaciones e instrucciones con el laboratorio. Ajueste fisiológicos del armazón y los procedimientos de impresión en la técnica del modelo alterado. Relaciones y registros maxilomandibulares. Desarrollo oclusal y estético. Principios y procedimientos de inserción. Preparación del paciente e instrucciones y cuidados posteriores. Procedimientos clínicos y de laboratorio. Métodos de laboratorio. Procedimientos clínicos