Assessment of Treatment-Relevant Immune Biomarkers in Psoriasis and Atopic Dermatitis: Toward Personalized Medicine in Dermatology.

Immunologically targeted therapies have revolutionized the treatment of inflammatory dermatoses, including atopic dermatitis and psoriasis. Although immunologic biomarkers hold great promise for personalized classification of skin disease and tailored therapy selection, there are no approved or widely used approaches for this in dermatology. This review summarizes the translational immunologic approaches to measuring treatment-relevant biomarkers in inflammatory skin conditions. Tape strip profiling, microneedle-based biomarker patches, molecular profiling from epidermal curettage, RNA in situ hybridization tissue staining, and single-cell RNA sequencing have been described. We discuss the advantages and limitations of each and open questions for the future of personalized medicine in inflammatory skin disease.

Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps.

The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway depends on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Polζ or Polη, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Polζ or Polη depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

Correction: Ma et al. Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2. Cells 2021, 10, 1467.

The authors wish to make the following changes to their paper [...].

Rad52 Oligomeric N-Terminal Domain Stabilizes Rad51 Nucleoprotein Filaments and Contributes to Their Protection against Srs2.

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation.

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

Correction: The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.

[This corrects the article DOI: 10.1371/journal.pgen.1007119.].

The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.

Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage.

Impact of bronchopulmonary dysplasia on brain and retina.

Many premature newborns develop bronchopulmonary dysplasia (BPD), a chronic lung disease resulting from prolonged mechanical ventilation and hyperoxia. BPD survivors typically suffer long-term injuries not only to the lungs, but also to the brain and retina. However, currently it is not clear whether the brain and retinal injuries in these newborns are related only to their prematurity, or also to BPD. We investigated whether the hyperoxia known to cause histologic changes in the lungs similar to BPD in an animal model also causes brain and retinal injuries. Sprague Dawley rat pups were exposed to hyperoxia (95% O2, 'BPD' group) or room air (21% O2, 'control' group) from postnatal day 4-14 (P4-14); the rat pups were housed in room air between P14 and P28. At P28, they were sacrificed, and their lungs, brain, and eyes were extracted. Hematoxylin and eosin staining was performed on lung and brain sections; retinas were stained with Toluidine Blue. Hyperoxia exposure resulted in an increased mean linear intercept in the lungs (P<0.0001). This increase was associated with a decrease in some brain structures [especially the whole-brain surface (P=0.02)], as well as a decrease in the thickness of the retinal layers [especially the total retina (P=0.0008)], compared to the room air control group. In addition, a significant negative relationship was observed between the lung structures and the brain (r=-0.49,P=0.02) and retina (r=-0.70,P=0.0008) structures. In conclusion, hyperoxia exposure impaired lung, brain, and retina structures. More severe lung injuries correlated with more severe brain and retinal injuries. This result suggests that the same animal model of chronic neonatal hyperoxia can be used to simultaneously study lung, brain and retinal injuries related to hyperoxia.

Rad52 sumoylation prevents the toxicity of unproductive Rad51 filaments independently of the anti-recombinase Srs2.

The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.

From simple bacterial and archaeal replicons to replication N/U-domains.

The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three domains of life. Here, we review our knowledge of genome organization into single and multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons show degenerate specificity and efficiency, allowing for complex regulation of origin firing time. We expand on recent evidence that ~50% of the human genome is organized as ~1,500 megabase-sized replication domains with a characteristic parabolic (U-shaped) replication timing profile and linear (N-shaped) gradient of replication fork polarity. These N/U-domains correspond to self-interacting segments of the chromatin fiber bordered by open chromatin zones and replicate by cascades of origin firing initiating at their borders and propagating to their center, possibly by fork-stimulated initiation. The conserved occurrence of this replication pattern in the germline of mammals has resulted over evolutionary times in the formation of megabase-sized domains with an N-shaped nucleotide compositional skew profile due to replication-associated mutational asymmetries. Overall, these results reveal an evolutionarily conserved but developmentally plastic organization of replication that is driving mammalian genome evolution.

Do replication forks control late origin firing in Saccharomyces cerevisiae?

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions.

Giant yeast cells with nonrecyclable ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle. rnr1C883A rnr3Δ cells grow under constant replicational stress, as evidenced by the constitutive activation of the checkpoint effector Rad53, and their S phase is considerably extended compared to the wild type. rnr1C883A rnr3Δ mutants also display additional abnormalities such as a median cell volume increased by a factor of 8, and the presence of massive inclusion bodies. However, they exhibit a good plating efficiency and can be propagated indefinitely. rnr1C883A rnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.

A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.

Visualization of ribonucleotide reductase catalytic oxidation establishes thioredoxins as its major reductants in yeast.

Thioredoxins and/or glutaredoxins assist ribonucleotide reductase, and other such enzymes that require disulfide bond reduction during their catalytic cycle. In Saccharomyces cerevisiae, the presence of either pathway is essential but which of these pathways operates in ribonucleotide reductase reduction and how this function contributes to the pathways' essential nature have not been definitively established. We have identified two in vivo redox forms of the S. cerevisiae ribonucleotide reductase R1 subunit, which correspond to catalytically reduced or oxidized enzymes. Cells lacking thioredoxins, which exhibit an elongated S phase, accumulate R1 in its oxidized form and also contain significantly decreased deoxyribonucleotide levels during the S phase. Overexpressing R1 in these cells increases both the amount of the R1 reduced form and the concentrations of deoxyribonucleotides and accelerates DNA replication. These results establish thioredoxins as the major RNR reducing system in yeast and indicate that impaired RNR reduction accounts for the S phase defects of thioredoxin-deficient cells.

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.

Mismatch tolerance by DNA polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae.

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3'-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.