bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using budding yeast to study properties and function of the human Replication Protein A2 N‐terminus in response to specific‐types of DNA damage
Authors: Haring Stuart J. , Senger Barbara L. , Wilson Timothy M. 
Published: November 2023

BackgroundReplication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus (NT) of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that phosphorylation of the yeast Rpa2 NT does not occur under commonly assayed genotoxic conditions, yet this domain is vital for the damage response. Using a chimeric yeast Rpa2 protein containing the human Rpa2 N‐terminus, we have demonstrated that this human domain is phosphorylated in yeast cells a manner nearly indistinguishable from that in human cells.Study ObjectiveWe wish to determine properties of human Rpa2 NT phosphorylation, including kinases required, sites involved and their interdependence, and what conditions trigger this phosphorylation to identify whether properties of Rpa2 NT phosphorylation are conserved in yeast and human cells.MethodsWe utilized chimeric human‐yeast Rpa2 proteins (with WT and mutant NT), yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for the human Rpa2 NT to be phosphorylated in response to varying DNA damaging agents.Results and ConclusionsWe determined that the same sites in the human Rpa2 NT are phosphorylated on the chimeric protein in yeast, and that these show similar dependence on DNA damage induction. Unlike that reported in human cells, phosphorylation of one site does not depend on phosphorylation of another site within the domain (i.e., no sequential/primed phosphorylation). We demonstrated that phosphorylation of the human Rpa2 NT in yeast only appears to occur when cells are treated with agents that produce double‐strand breaks (e.g., HO endonuclease, phleomycin, camptothecin) but not when replication is inhibited by hydroxyurea. Finally, we have shown that Mec1 (yeast ATR), but surprisingly not Tel1 (yeast ATM), is necessary to phosphorylate T21 and S33 (both containing TQ or SQ) on the human Rpa2 NT. This is in contrast to human cells where ATR has only been reported to phosphorylate S33, not T21. We conclude that yeast provides a simple system to identify kinases involved, conditions that trigger human Rpa2 phosphorylation, and its relation to other properties of damage responses. This also provides the opportunity to study Rpa2 NT modification in two different systems to gain an understanding of conserved properties and to potentially study the significance of differences in the damage response between the two systems.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH.
Protein‐protein interaction inhibitors of the Fanconi anemia DNA repair pathway
Authors: Voter Andrew F. , Manthei Kelly A. , Keck James L. 
Published: November 2023

Upregulation of the Fanconi anemia DNA repair pathway is a frequent mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Previous studies have determined that the interaction between FANCM and the RecQ mediated genome instability (RMI) complex is essential for crosslink repair in this pathway and that disruption of the interface causes sensitization to DNA crosslinking agents. To identify small molecules that disrupt this interaction, we developed a high‐throughput fluorescence polarization assay that was used to screen a library of 75,000 small molecules. We confirmed the activity of a selective inhibitor in an orthogonal proximity assay and detected its direct physical interaction with the RMI complex using surface plasmon resonance and isothermal titration calorimetry. More potent inhibitors have been identified by structure activity relationship studies. These potent small molecules will be tested for synergies with DNA crosslinking agents in vivo to assess for therapeutic potential.Support or Funding InformationUniversity of Wisconsin – Madison Medical Scientist Training Program (T32GM008692), Integrated Program in Biochemistry, ICTR Clinical and Translational Science award (UL1TR00427) and traineeship (TL1TR000429) and the NCI (R21CA178475‐01)
DNA Cytosine Methyltransferase Increases Stationary Phase Fitness under Temperature Stress in Escherichia coli
Authors: Huss Rebecca A , Davidson Samantha , Militello Kevin T 
Published: November 2023

Escherichia coli contains two orphan DNA methyltransferases. DNA adenine methyltransferase (Dam) methylates 5′GATC3′ sequences, and DNA cytosine methyltransferase (Dcm) methylates the interior cytosine in 5′CCWGG3′ sequences. Numerous functions of the Dam enzyme have been elucidated, however the role of Dcm is less defined. Growth curve assays in our laboratory have shown no significant difference in growth rate between strains containing dcm and those without it. However, our laboratory's DNA microarray analyses have revealed numerous gene expression changes in dcm knockout cells in early stationary phase, suggesting dcm influences stationary phase biology. Thus, we tested the role of Dcm in stationary phase fitness. A dcm containing plasmid was constructed by amplifying the dcm gene as well as the putative promoter upstream of the gene by PCR, and inserting it into the pET‐28a+ plasmid. The pET‐28a+ plasmid with and without dcm were introduced into T7 express bacteria, which has a large chromosomal deletion that includes the dcm gene. We call the new strains T7E/pDcm and T7E/pET‐28a+. In order to demonstrate that the cells containing dcm methylated their DNA, a restriction enzyme isoschizomer assay was conducted with PspGI and BstnI. The results indicated that the cells containing T7E/pDcm methylated their DNA at 5′CCWGG3′ sites. The T7E/pET‐28a+ and T7E/pDcm strains were competed over long‐term stationary phase at 37°C. Colony PCR assays were used to determine which strain was outcompeting the other at the conclusion of one and three month intervals. At 37°C, T7E/pET‐28a+ outcompeted T7E/pDcm. As our laboratory has revealed in recent microarray studies, the cold‐shock protein B gene (cspb) was upregulated in bacteria treated with the DNA methyltransferase inhibitor, 5‐azacytidine. This suggests a possible biological role for Dcm at colder temperatures. We then competed T7E/pET‐28a+ and T7E/pDcm at 20°C over long‐term stationary phase. When competing at 20°C, T7E/pDcm outcompeted T7E/pET‐28a+. In order to verify the surviving T7E/pDcm colonies still methylated their DNA, restriction enzyme isoschizomer assays were performed, with the results indicating methylation occurs in only the T7E/pDcm colonies. Our work suggests a new and unexpected phenotype for Dcm. Dcm provides a fitness advantage during stationary phase at low temperatures. We aim to determine the biological mechanism for this phenotype.Support or Funding InformationThis work was supported by the Geneseo Foundation.
Identifying the Molecular Mechanisms by which Disruptor of Telomere silencing 1‐Like (DOT1L), a Histone 3, Lysine 79 (H3K79) Methyltransferase, Regulates Mammalian Hematopoiesis
Authors: Malcom Carrie , Piasecka‐Srader Joanna , Alvarez Nehemiah , Slawson Chad , Fields Patrick 
Published: November 2023

Disruptor of Telomere silencing 1‐Like (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase. The enzyme has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. Previous studies in our lab found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid‐gestation (Feng et al., 2010). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. Specifically, we sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis. DOT1L is a large protein (1540aa) and involved in several, diverse processes, but only its methyltransferase activity has been documented. Additional functional domains of the protein might be responsible for its diverse activities.We developed a murine embryonic stem cell (mESC)‐based culture system in which to examine the role of DOT1L in hematopoiesis, in vitro. Using the Cas9/CRISPR system, we created two different mutations in Dot1L in cultured mESCs: a Dot1L knockout and a Dot1L point mutant. Knockout mESCs contain a large, out‐of‐frame deletion in both alleles of Dot1L, while the point mutant contains a single amino acid change in the methyltransferase domain of Dot1L, thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We then induced these Dot1L knockout and point mutant mESC clones, along with wildtype mESCs, to undergo hematopoiesis in culture. Our data showed that this protocol leads to similar erythropoietic defects as those observed in vivo. Dot1L knockout and point mutant mESCs are capable of forming the same types of hematopoietic colonies as wildtype mESCs (both erythroid and myeloid), but there is a decrease in colony size and number compared to wildtype. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is entirely responsible for its function in facilitating early, murine hematopoiesis.Support or Funding InformationR01 DK091277, Fields (PI), 04/01/12‐03/31/17NIH/NIDDKRole of the Histone Methyltransferase DOT1L in Embryonic Erythropoiesis
Quantitative Histone H3 Modification Characterization of Differentiating Hematopoetic Murine Erythroleukemia (MEL) Cells Using Parallel Reaction Monitoring Mass Spectrometry
Authors: Hess Sonja , Moradian Annie , Raedle Matthias , Sweredoski Michael 
Published: November 2023

Murine Erythroleukemia cells differentiate into hematopoetic cells that produce either embryonic or adult hemoglobin, dependent on the chosen inducer (histone deacetylase inhibitor butyric acid vs. DMSO). The differentiation has, at least in part, been associated with changes in histone modifications that are not readily discernible by classic biochemical analyses such as antibodies or classical bottom‐up mass spectrometry.To establish a quantitative method for histone modifications, we developed a middle‐down proteomics workflow with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion.We then monitored the twenty most abundant combinations of methylation and acetylation of the N‐terminal tail of histone H3. The N‐terminal H3 tail was separated primarily by its acetylation states using weak cation exchange hydrophilic interaction chromatography. In addition, some separation can also be achieved by its methylation states.After extensive data evaluation, a minimum set of over 250 H3 proteoforms was identified. More importantly, quantitative acetylation and methylation maps were created. MEL cells treated with HDAC inhibitor butyric acid showed a bimodal distribution with one cluster showing significantly more acetylations and methylations than untreated samples and samples treated with DMSO. A second cluster emerged from the butryic acid treated samples showing significantly less acetylations and methylations than untreated and DMSO‐treated samples.Furthermore, we developed a fragment ion map to visualize specific differences between treated and untreated samples.Taken together the data presented here show that middle‐down proteomics with electron transfer dissociation using PRM is a novel, attractive mass spectrometry method for the effective analysis and quantification of histone tails.Support or Funding Information

This project was supported by the Gordon and Betty Moore Foundation through grant GBM275, the Beckman Institute, HHMI (Fusion) and UO1 grant 4DNucleome.
Analysis of Long Noncoding RNA Chromatin Interactions by Chromatin Isolation by RNA Purification (ChIRP)
Authors: Asbrock Nick 
Published: November 2023

Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non‐coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored.Chromatin Isolation by RNA Purification (ChIRP) is a method that allows discovery of the sites of interaction of chromatin‐associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe‐based hybridization to target RNA molecules in chromatin. DNA can be isolated from recovered chromatin and analyzed by quantitative PCR or next generation sequencing (ChIRP‐seq). Alternatively, RNA may also be isolated to detect other RNA molecules that may be associated with the RNA of interest.To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19) and peaks were called. We successfully identified several NEAT1 binding sites in the genomic DNA sequence.In summary the methods developed allow unbiased discovery of RNA‐associated genomic DNA sequences, RNA sequences, and potentially proteins.
The role of histone variant H2A.Z in co‐transcriptional splicing in Saccharomyces cerevisiae
Authors: Neves Lauren Taylor , Douglass Stephen , Galivanche Anoop , Johnson Tracy 
Published: November 2023

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre‐messenger RNA (pre‐mRNA). Although RNA‐synthesis and RNA‐processing have typically been studied as biochemically distinct reactions, recent studies have shown these two processes to be spatio‐temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited in to the chromatin by the SWR1‐complex and is typically found near the 5′ ends of protein‐coding genes. H2A.Z has been implicated in transcription elongation and polymerase processivity. Here we show that H2A.Z, encoded by HTZ1 in yeast, is important for efficient splicing of intron‐containing genes, and splicing in cells lacking H2A.Z is impaired under suboptimal conditions, such as mutant splicing complexes or suboptimal splice sites. HTZ1, is particularly dependent on an intact U2 snRNP, as it shows extensive genetic interactions with components of the U2 snRNP and U2 snRNP associated proteins. Consistent with this, H2A.Z is required for the splicing of introns containing a mutated splice site. RNA‐seq reveals that intron‐containing genes with non‐consensus branchpoint sequences are particularly sensitive to loss of H2A.Z. We find that deletion of HTZ1 results in persistent association of the U2 snRNP with nascent RNA, suggesting altered spliceosomal rearrangements in the absence of H2A.Z. Together, these results demonstrate that histone variant H2A.Z is required for efficient splicing of a subset of intron‐containing genes and indicate a role for H2A.Z in co‐transcriptional spliceosome assembly.Support or Funding InformationRuth L. Kirschstein National Research Service Award GM007185.
Discovery of mammalian regulators of ribosome biogenesis
Authors: Farley Katherine , McCann Kathleen , Merkel Janie , Surovtseva Yulia , Baserga Susan J. 
Published: November 2023

It is well known that in cancer an increased size and number of nucleoli correlates with increased malignant potential. Nucleoli make ribosomes, the essential protein synthesizers of the cell. Previously we have shown (PLoS Genetics 2012) that disruption of nucleolar function by depletion of the ribosome biogenesis factors UTP4 or NOL11 caused a change in the number of nucleoli per cell from 2–3 to only 1. Taking advantage of this relationship between number and function, we exploited the changes in nucleolar number to discover new regulators of nucleolar function in human cells via a high‐content, high‐throughput, genome‐wide siRNA screen. This screen successfully identified approximately 200 proteins whose depletion caused a change in the number of nucleoli per cell from 2–3 to only 1. The function of a subset of these hits in ribosome biogenesis has been validated by oligonucleotide deconvolution. Of these high‐confidence, validated hits, 22 were chosen for further study, including both nucleolar and non‐nucleolar proteins. While the nucleolar proteins are directly involved in ribosome biogenesis, the non‐nucleolar proteins likely reflect new pathways of nucleolar regulation in mammalian cells. Out of the 22 tested hits, we have identified new roles for 20 hits in regulating either the RNA polymerase I transcription of the ribosomal DNA (8/22), the processing of the pre‐ribosomal RNA (19/22), or in the maintenance of the ribosomal DNA chromatin state (2/22). These hits increase the complexity of ribosome biogenesis, extending it beyond the nucleolus to extra‐nucleolar protein regulators of nucleolar function.Support or Funding InformationNIGMS
Chemical Ligation Synthesis of Isotope‐Labeling Compatible Selenoprotein S
Authors: Subramoney Jay , Zhang Zhengqi , Cheng Rujin , Rozovsky Sharon 
Published: November 2023

Selenoproteins contain the 21st amino acid selenocysteine (Sec) and play vital roles in human health, such as antioxidant defense. Although selenoproteins are expressed naturally in the human body, procuring sufficient quantities for biochemical and biophysical characterization by heterologous expression is challenging. Low yield in genetic incorporation is due to the Sec codon being identical to the UGA stop codon in mRNA. An alternative approach for their preparation is chemical ligation, a semi‐synthetic method that relies upon an amide bond formation to generate peptides and proteins from their complementary fragments. Here, we describe a new method for chemical ligation of selenoprotein S (SelS) which is compatible with simple and low cost isotopic labeling and does not rely on complete chemical synthesis of a selenoprotein. SelS is a member of the endoplasmic reticulum associated protein degradation (ERAD) pathway, a protein machinery devoted to relocating misfolded proteins from the ER to a cytoplasmic protein degradation complex. The active site lies close to the C‐terminal where a sole Sec is paired with a Cysteine 13 residues away. Previously we have characterized the activity of SelS prepared by heterologous expression, however, structural characterization with the Sec in place was prohibited by the low yield. In our approach a Sec‐containing fragment is expressed in E. coli and used as a source of the selenopeptide during the chemical ligation. This has the advantage of a high yield, with simple and efficient ligation while being compatible with isotopic‐labeling of selenoproteins. Useful applications of this method include incorporation of Selenium‐77 and Carbon‐13 for structural and biochemical characterization by NMR spectroscopy.Support or Funding InformationThis work was funded by the National Institute of General Medical Science
The PINK‐Parkin Circuit in Mitochondrial Quality Control
Authors: Liu Xuedong , Zhang Conggang , Peng Yinghua , Bunker Eric , Lee Schuyler 
Published: November 2023

Mutations in E3 ubiquitin ligase Parkin and Ser/Thr protein kinase Pink1 cause autosomally inherited parkinsonism. Genetic studies have shown that Pink1 and Parkin act in the same pathway that regulates mitochondria dynamics, motility, and quality control. Activation of this pathway can be triggered by a variety of cellular stressor signals that cause mitochondrial damage.Here, we present evidence that PINK1‐Parkin has both cytoprotective and pro‐apoptotic functions. PINK1‐Parkin operates as a molecular switch to dictate cell fate decisions in response to different cellular stressors. Cells exposed to severe and irreparable mitochondrial damage agents such as valinomycin can undergo PINK1‐Parkin‐dependent apoptosis. The proapoptotic response elicited by valinomycin is associated with the degradation of Mcl‐1. PINK1 directly phosphorylates Parkin at Ser65 of its Ubl domain and triggers activation of its E3 ligase activity through an autocatalytic mechanism which amplifies its E3 ligase activity towards Mcl‐1. Autocatalytic activation of Parkin bolsters it accumulation on mitochondria and apoptotic response to valinomycin. Our results suggest that PINK1‐Parkin constitutes a damage‐gated molecular switch that governs cellular context‐specific cell fate decisions in response to variable stress stimuli. We developed an analog sensitive PINK1 allele and demonstrated that PINK1 is required for triggering Parkin translocation to mitochondria, but is dispensable for sustaining Parkin accumulation and consequent mitophagy.Support or Funding InformationThis work was supported by grants from a Butcher Award from the University of Colorado and National Institutes of Health CA107098 and GM113141 to Xuedong Liu. The ImageXpress MicroXL was supported by a NCRR grant S10 RR026680 from NIH.
Adaptor Guided Proteolysis Initiates on the Ribosome
Authors: Puri Neha , Woo Perry , Mehta Preeti , Choksi Arnav , Karzai Wali 
Published: November 2023

ObjectiveTo characterize the capture of tmRNA‐tagged proteins by the SspB adaptor on the ribosomes.MethodsWe utilized in vitro enzyme kinetics and in vivo ribosome enrichment assays to study SspB‐ribosome interactions and the consequential degradation of tagged protein. We isolate total ribosomes and determine the relative amounts of SspB associated with the ribosomes by specifically enriching for ribosomes translating the reporter mRNA of choice.ResultsAdaptor proteins extend the substrate range and specificity of AAA+ proteases. The SspB adaptor protein enhances the efficiency of ClpXP mediated proteolysis of tmRNA‐tagged proteins. Our investigations into whether ribosomes serve as a hub for initiating directed proteolysis reveal that SspB captures tag‐bearing proteins on tmRNA‐recued ribosomes in a trans‐translation dependent manner, in a process reliant on distinct recognition signals within the tmRNA degron. Defined alterations in the SspB and ClpX recognition elements of the tmRNA peptide sequence, or the peptide‐binding groove of SspB, have adverse effects on the efficiency of SspB ribosome recruitment and degradation of marked proteins.ConclusionsOur studies suggest that SspB scans translating ribosomes to capture tmRNA tagged proteins and guide their delivery to ClpXP for directed proteolysis. Our inquiries have also unveiled the capacity of SspB to capture additional nascent polypeptides that harbor genetically encoded yet degenerate forms of the tmRNA degron and modulate their intracellular abundance through selective proteolysis.Support or Funding InformationNIH