SUMO is a pervasive regulator of meiosis

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here, we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Comparison of ERCC1/XPF genetic variation, mRNA and protein levels in women with advanced stage ovarian cancer treated with intraperitoneal platinum.

OBJECTIVE: Approximately 20% of patients receiving platinum-based chemotherapy for epithelial ovarian cancer (EOC) are refractory or develop early recurrence. Identifying these patients early could reduce treatment-associated morbidity and allow quicker transfer to more effective therapies. Much attention has focused on ERCC1 as a potential predictor of response to therapy because of its essential role in the repair of platinum-induced DNA damage. The purpose of this study was to accurately measure protein levels of ERCC1 and its essential binding partner XPF from patients with EOC treated with platinum-based therapy and determine if protein levels correlate with mRNA levels, patient genotypes or clinical outcomes. METHODS: ERCC1 and XPF mRNA and protein levels were measured in frozen EOC specimens from 41 patients receiving intraperitoneal platinum-based chemotherapy using reverse transcription polymerase chain reaction and western blots. Genotypes of common nucleotide polymorphisms were also analyzed. Patient outcomes included progression free (PFS) and overall survival (OS). RESULTS: Expression of ERCC1 and XPF were tightly correlated with one another at both the mRNA and protein level. However, the mRNA and protein levels of ERCC1 were not positively correlated. Likewise, none of the SNPs analyzed correlated with ERCC1 or XPF protein levels. There was an inverse correlation between mRNA levels and patient outcomes. CONCLUSION: Neither genotype nor mRNA levels are predictive of protein expression. Despite this, low ERCC1 mRNA significantly correlated with improved PFS and OS

2,4,6-Trimethoxy chalcone derivatives: an integrated study for redesigning novel chemical entities as anticancer agents through QSAR, molecular docking, ADMET prediction, and computational simulation

QSAR, an efficient and successful approach for optimizing lead compounds in drug design, was employed to study a reported series of compounds derived from 2,4,6-trimethoxy chalcone derivatives. The ability of these compounds to inhibit CDK1 was examined, with the help of QSARINS software for model development. The generated QSAR model revealed three significant descriptors, exhibiting strong correlations with impressive statistical values: cross-validation leave-one-out correlation coefficient (Q2LOO) = 0.6663, coefficient of determination (R2) = 0.7863, external validation coefficient (R2ext) = 0.7854, cross-validation leave-many-out correlation coefficient (Q2LMO) = 0.6256, Concordance Correlation Coefficient for cross-validation (CCCcv) = 0.8150, CCCtr = 0.8804, and CCCext = 0.8750. From the key structural findings and the insights gained from the descriptors, ETA_dPsi_A, WTPT-5, and GATS7s, new lead molecules were designed. The designed molecules were then evaluated for their CDK1 inhibitory activity using the three-descriptor model developed in this study. To evaluate their drug likeliness, in-silico ADMET predictions were made using Schrodinger’s Software. Molecular docking was carried out to determine the interactions of designed compounds with the target protein. The designed compounds having excellent binding pocket molecular stability and anticancer effectiveness was substantiated by the findings of the molecular dynamics simulation. The results of this work point out important properties and crucial interactions necessary for efficient protein inhibition, suggesting lead candidates for further development as novel anticancer agents. Communicated by Ramaswamy H. Sarma</p

Correction of <i>XPF</i> mutant cell NER defect by microinjection of XPF-ERCC1.

<p>Primary fibroblasts from XFE progeroid patient XP51RO were fused to create homopolykaryons by treatment with inactivated Sendai virus then plated on glass coverslips. Only homopolykaryons were injected with recombinant XPF-ERCC1 protein complex (A) wild-type (B) XPF^R799W-ERCC1 (C) XPF^R153P-ERCC1. The cultures were irradiated with 10 J/m² UV-C and ³H-thymidine was added to the culture. UV-induced unscheduled DNA synthesis was detected by autoradiography. Homopolykaryons are indicated with arrows. (D) Histogram indicating the average number of radiographic grains in nuclei injected with each of the recombinant protein complexes and uninjected cells in the same sample. Error bars indicate the standard deviation. N indicates the number of nuclei analyzed in each population. P values for the comparison between injected and uninjected cells were calculated using an unpaired two-tailed Student's t-test.</p

Characteristics of <i>XPF</i> mutant cell lines.

<p>UDS unscheduled DNA synthesis</p><p>*The patient had normal levels of <i>XPF</i> transcript, suggesting one allele encodes a full-length mRNA.</p>♦<p>Mutation could not be confirmed on genomic DNA.</p><p>° Siblings.</p><p>n.d.  =  not determined</p

Characterization of XPF-YFP and XPF¹⁵³-YFP in CHO cells.

<p>(A) Western blot analysis of XPF-YFP expressed in <i>Xpf</i> mutant cells. XPF-deficient hamster cell line, UV41, was transiently transfected with wild type <i>XPF-YFP</i> or <i>XPF¹⁵³-YFP</i> and the fusion proteins were detected using an antibody against XPF or GFP. C5RO was used as positive control for the XPF blot and as a negative control for the GFP blot. UV41 cells transfected with YFP alone was used as a negative control for XPF blot and as a positive control for GFP blot. (B) Clonogenic survival of wild-type (wt), XPF-deficient CHO cell line UV41, and UV41 transfected with wild type XPF-YFP and XPF¹⁵³-YFP after UV and MMC treatment. Colonies were counted 7–10 days after treatment and results are plotted as mean 3 independent experiments. (C) Subcellular localization of wild type XPF-YFP and XPF¹⁵³-YFP after transient transfection in XPF-deficient the CHO cell line UV41 detected by fluorescence microscopy.</p

Biochemical characterization of XPF^R153P-ERCC1 and XPF^R799W-ERCC1 mutants.

<p>(A) Gel filtration profiles from the purification of recombinant XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1 from baculovirus-infected Sf9 insect cells using a His₆ tag on ERCC1. Aggregated proteins elute at ∼45 ml in the void volume of the column; heterodimeric XPF-ERCC1 elutes at ∼65 ml, corresponding to ∼200 kD, and monomeric ERCC1 elutes at ∼78 ml (∼50kD). (B) SDS PAGE analysis of purified protein complexes. Lane 1, 3 and 5 (D): XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1, respectively, after purification over NTA-agarose, gel filtration and heparin columns. Lanes 2 and 4 (A) show the proteins present in the fractions eluting at 45 ml in the gel filtration column step of XPF^R153P-ERCC1 and XPF^R799W-ERCC1, respectively. (C) Immunodetection of XPF in normal (C5RO) and <i>XPF</i> mutant cells. The star indicates the migration of a cross-reactive band demonstrating equal loading <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000871#pgen.1000871-Niedernhofer2" target="_blank">[32]</a>. (D) Incision activities of XPF-ERCC1, XPF^R153P-ERCC1 and XPF^R799W-ERCC1 (200 fmol) on a 5′-³²P-labeled stem-loop DNA substrate (100 fmol) in the presence of either 0.4 mM MnCl₂ (lanes 2, 4 and 6) or 2 mM MgCl₂ (lanes 3, 5 and 7). Reactions were analyzed on a 15% denaturing polyacrylamide gel. The 46-mer substrate and 9–10-mer products are indicated.</p