bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potent DNA Strand Annealing Mediated by the T7 Single‐Stranded DNA Binding Protein gp2.5
Authors: Hernandez Alfredo J. , Richardson Charles C. 
Published: November 2023

Single‐stranded DNA binding proteins are ubiquitous in all domains of life and play essential roles in DNA replication, recombination, and repair. Bacteriophage T7 encodes a single‐stranded DNA binding protein, gp2.5, which is crucial for coordinating the synthesis of leading and lagging DNA strands.In addition to its role in DNA replication, T7 gp2.5 displays a potent homologous DNA strand annealing activity, which is necessary for genetic recombination. Gp2.5 enhances the annealing of DNA strands orders of magnitude over the spontaneous rate, and is considerably more efficient compared to other recombination proteins. The annealing of complimentary strands by gp2.5 is strictly specific for DNA. While the annealing reaction is somewhat tolerant to internal mismatches, it is very permissive to internal, unpaired loops.Here we provide a model for the mechanism of gp2.5‐mediated homologous strand annealing and explore its applicability in oligonucleotide‐mediated gene modification and the formation of higher‐order DNA structures in vitro.
Y‐family DNA Polymerase Human Pol Kappa Is More Tolerant of Changes to Its Active Site Loop Than DinB
Authors: Antczak Nicole M , Packer Morgan , Walsh Jason , Ippoliti Paul , Beuning Penny J 
Published: November 2023

DNA damage is a constant threat from both endogenous and exogenous sources. Exposure to UV radiation and reactive oxygen species can cause DNA damage. Most damage undergoes a repair process but damage that is not repaired is bypassed in a process called translesion synthesis. Y‐family DNA polymerases carry out translesion synthesis by replicating damaged DNA as they have a larger active site to accommodate bulky DNA adducts. Y‐family DNA polymerases are conserved in all domains of life. In E. coli, there are two, DinB (Pol IV) and UmuD′2 (Pol V), one in the archaeon Sulfolobus solfataricus, Dpo4, and several in humans including pol kappa, an ortholog of DinB. Both DinB and pol kappa have been shown to bypass minor groove adducts on the N2 position of deoxyguanine while Dpo4 can bypass both minor groove and major groove adducts. Although DinB and pol kappa are homologous, we have previously shown they are blocked to different extents by major groove lesions etheno‐dA and N6‐furfuryl‐dA and that a single point mutation in an active site loop in DinB eliminates discrimination against N6‐furfuryl‐dA.In this work we created chimeric polymerases by swapping the loop region adjacent to the active site using site directed mutagenesis to assess the importance of the loop in damage specificity and activity. Using primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adducts etheno‐dA and N6‐furfuryl‐dA, as well as undamaged templates, we found the loop regions of DinB and pol kappa near the catalytic site are important for damage bypass specificity and accuracy of nucleotide incorporation. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of major groove adducts by DinB and hPol κ can be accomplished.Support or Funding InformationSupport from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.
Role of Pol κ and DinB distal residues on extension step of TLS
Authors: Stern Hannah R , Mills Caitlyn L , Ondrechen Mary Jo , Beuning Penny J 
Published: November 2023

DNA is subject to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass lesions when copying damaged DNA in a process called translesion synthesis. DinB is one of these polymerases, which is involved in bypassing deoxyguanosine adducts at the N2 position. There are also four human Y family polymerases, including DNA polymerase Kappa, that have similar function. However, Pol κ is more efficient in copying past DNA damage lesions in the extension step of translesion synthesis. In order to investigate the importance of particular residues in the extension step of TLS, the computational method POOL was utilized. This tool identified active site residues and residues previously observed to be important for activity. POOL also predicted more distant residues that do not have direct contact with substrate that may have catalytic importance, but the residues are in different regions of DinB and Pol κ. In addition, previous HXMS work done in our lab suggested a possible allosteric change in both DinB and Pol κ in different distal residues that POOL did not predict. To study the contribution of these distal residues on the extension step of TLS, DinB and Pol κ variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage lesions.Support or Funding InformationSupport from NSF‐MCB‐1517290, American Cancer Society RSG‐12‐161‐01‐DMC, and the PhRMA Foundation (predoctoral fellowship in informatics awarded to CLM)
Investigating the mechanism of trans‐lesion synthesis by human DNA polymerase kappa
Authors: Allen Taylor Brooke , Younger Zane , Benitez Benedetta Sampoli 
Published: November 2023

Our DNA is constantly under attack by both exogenous and endogenous chemicals that have the ability to damage it. If not repaired, these DNA adducts can lead to mutations to the DNA or even worse, they can halt replication, eventually leading to aging and/or cancer. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases, known for their ability to conduct trans‐lesion synthesis of damaged DNA. So far, the mechanism for lesion bypass discrimination by pol k is unclear. Recent studies suggested that similar to other polymerases, pol k might cycle between different open and closed conformations upon binding to DNA and the substrate. Through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotides. In particular, we simulated a binary system, with DNA and protein only, ternary systems with correct and incorrect incoming nucleotides, and a ternary system with with a O6‐methylated guanine (O‐metG) on the templating strand, a damage which pol k cannot bypass. Preliminary results show that pol k does not attain a close, catalytically competent state both in the binary and the O‐metG system, as evidenced by the few contacts between the protein and the incoming nucleotide. We have also identified key residues important for the stabilization of the incoming nucleotide as well as a hypothesis to why the O‐metG does not get bypassed as efficiently as other damages.Support or Funding InformationThis work was funded by the Rose M. Badgeley Residuary Charitable Trust and the Sister Colette Mahoney Science Research Grant.
INO80 Chromatin Remodeling Connects Metabolic Gene Expression to Cell Division
Authors: Morrison Ashby J , Gowans Graeme , Schep Alicia , King Devin , Greenleaf William 
Published: November 2023

The coordination of cellular function with the environment is essential for adaptation and survival. S. cerevisiae has a remarkable ability to sense diverse (i.e. nutrient‐rich or □limiting) environments and reprogram their energy metabolism and proliferative capacity accordingly. These adaptive cellular responses are often achieved by rapid inducible changes in metabolic gene expression facilitated by chromatin modification.Previously we identified the INO80 chromatin remodeler as a primary regulator of gene expression in energy metabolism pathways (Yao et al, MCB 2016). We then sought to examine the role of INO80 in coordinating metabolic homeostasis with cell division in metabolically synchronized yeast. Synchronization is achieved through nutrient depletion of yeast cultures followed by constant perfusion of glucose. Subsequently, the Yeast Metabolic Cycle (YMC) is induced, characterized by continuous respiration cycles of oxygen consumption. Within the YMC, most genes are periodically expressed in specific phases of quiescence, cell growth, and cell division. However, little is known about how these genes are so precisely regulated.We find that the INO80 complex is critical for organizing global gene expression and chromatin organization in the YMC. Resulting cellular phenotypes include altered respiration cycles and loss of metabolic coordination with cell division. Thus, the INO80 chromatin‐remodeler is central regulator of connecting metabolic gene expression to cell division.Support or Funding InformationNational Institutes of Health; R35 GM119580
Biochemical Insights into the Mechanism of Oncohistones
Authors: Lewis Peter W 
Published: November 2023

Recently, genome‐wide sequencing technologies have allowed the notable discovery of somatic mutations in chromatin modifying factors in many types of cancers. The collective number of oncogenic mutations has led to the emerging view of “driver mutations” in chromatin regulators underlying many human malignancies. Specific pediatric brain and bone tumors are reported to harbor a remarkably high frequency of monoallelic, gain‐of‐function mutations in genes encoding canonical (H3.1/2) or variant (H3.3) histone H3. These high frequency mutant histones (collectively referred to as ‘oncohistones’) are suspected drivers of specific types of human cancer. Previously, we demonstrated that lysine‐to‐methionine (K‐to‐M) substitutions transform histone H3 proteins from substrates into potent and highly specific inhibitors of SET domain‐containing lysine methyltransferase enzymes (e.g. K27M found in ~80% of midline gliomas, and K36M found in ~95% of chondroblastomas) and affect overall levels of histone modifications. We find that that the cofactor S‐adenosyl methionine (SAM) is necessary for stable interaction between SET domain enzymes and K‐to‐M oncohistones. Consistent with the formation of a ternary complex, we find that oncohistones are uncompetitive inhibitors with regard to SAM.Given the restricted distribution of H3 mutations in human cancers, we hypothesize that cell lineage is crucial for the ability of oncohistones to promote tumorigenesis. Therefore, we are using a combination of biochemical investigation, accurate model generation, and study of human tumor samples to comprehensively understand how H3 mutations mediate tumorigenesis. We find that the H3 K36M mutant histone impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. Depleting H3K36 methyltransferases, or expressing an H3 K36I mutant that similarly inhibits H3K36 methyltransferases, is sufficient to phenocopy the H3 K36M mutation. Upon loss of H3K36 methylation, a genome‐wide gain in H3K27 methylation leads to a redistribution of PRC1 and de‐repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified. I will discuss our recent work to characterize how oncohistones perturb cellular differentiation through inhibition of specific chromatin‐modifying enzymes.
Free hemoglobin change gene expression involving in cell‐cell signaling through different DNA methylation in THP‐1‐derived macrophages
Authors: Cha Min Ho , Lee Myeong Soo 
Published: November 2023

BackgroundFree hemoglobin formed from hemolysis of red blood cell, was known a predicator of many disease such as cancer, sclerosis and delirium. That also related with cytokine production in leukocytes. In this study, we elucidated change of gene expression through DNA methylation difference by free hemoglobin in THP‐1 derived macrophage.MethodsPMA induced THP‐1 derived macrophage were treated 50ug/ml of free hemoglobin (purchased from Sigma). DNA methylation were analyzed through whole genome bisulfate sequencing (WGBS) using TruSeq® DNA Methylation Kit (illumina) and 을 Illumina HiSeq. platform (illumina). Sequence quality were checked by FastQC and Trimmomatic and data analyzed by BSMAP. Difference of DNA methylation were determined in delta‐mean>[0.75] and p <0.05). Expression of genes were determined by reverse transcriptional PCR (RT‐QCR) and q‐PCR.ResultsDNA methylation of 29,706 CpG sites in total genome and 553 CpG sites in promoter region were changed by hemoglobin. Among 553 CpG sites in promoters, 267 CpG sites were hypomethylated and 286 sites were hypermethylated. GO annotation showed genes involving in neurological system process, ion transport, cell‐cell signaling and skeletal system development were significantly affected by hemoglobin. Genes involved in p53 cardiovascular system, cell adhesion molecules were significantly affected. Among of them, we focused 23 genes, which were included in cell‐cell signaling, and checked gene expression of five genes. IL‐7 and PCDN1 showed significant change of gene expression.DiscussionOur results showed that increase of free hemoglobin affects gene expression through change of DNA methylation and these might suggest that free hemoglobin level in vessel should be affect cell‐cell signaling and free hemoglobin might predictor of some diseases.Support or Funding InformationThis research was supported by a Grant from the Korea Institute of Oriental Medicine (C16111) and funds from Korea Food Research Institute (G16020)
Upregulating Human Leukocyte Antigen Expression in Multiple Cancer Cells through Epigenetic Modulators
Authors: Terrigino Nicholas Taylor , Nicholas David Joseph , Powers Rachel Elizabeth , Dutta Shourik , O'Donnell Robert Watts 
Published: November 2023

This study attempts to discern whether two epigenetic modulators, 5‐Azacytidine and Entinostat, increase the expression of Human Leukocyte Antigen (HLA) proteins on human cancer cells when used in combination. HLA is routinely down‐regulated in cancer, presumably to escape immunologic detection. We have previously shown that 5‐Azacytidine, a cytosine DNA methyltransferase inhibitor, increased HLA expression. Others have shown that Entinostat, an epigenetic modulator that functions as a histone deacetylase inhibitor, can modify tumor growth both in vivo and in vitro. Five cancer cell lines, HL‐60, MDA‐MB‐435, MDA‐MB‐231, MCF‐7, and HTB‐4 cells are being tested using flow cytometry and Phycoerythrin (PE) conjugated antibodies against the HLA Class I antigens A, B and C. Initial findings show that Entinostat, and/or the combination of Entinostat and 5‐Azacytidine, are more effective at up‐regulating HLA expression than 5‐Azacytidine alone in HL‐60, HTB‐4, and MDA‐MB‐435 cell lines. Further cells lines will be tested to determine if Entinostat or the combination will be more effective in up‐regulating HLA expression as well as the optimal dose and exposure time. If 5‐Azacytidine and Entinostat are shown to work in synergy, cancer cells could become better targets for cancer immunotherapy.Support or Funding InformationGeneseo Foundation and the SUNY Geneseo Biology Department
A Shared Structural Recognition Element in mRNA Substrates of the tRNA Modifying Enzyme Pseudouridine Synthase 1
Authors: Carlile Thomas M , Bell Tristan A. , Rojas‐Duran Maria F , Zinshteyn Boris , Shin Hakyung , Mason Caitlyn , Gilbert Wendy V 
Published: November 2023

RNA is extensively chemically modified in all organisms. These modifications were thought to be restricted to stable non‐coding RNAs, however recent work has revealed the presence of a variety of post‐transcriptional modifications in mRNA. Our lab recently developed Pseudo‐seq, a transcriptome‐wide approach to pseudouridine identification, which led to the discovery of widespread pseudouridylation of mRNAs in yeast, mouse, and human cells. We found that mRNA pseudouridylation events are regulated in response to cellular growth state; however, little is known about the mechanisms responsible for selective, regulated targeting of specific sequences for modification. We have sought to understand the mechanisms underlying how the conserved pseudouridine synthase Pus1 recognizes its mRNA targets. We have reconstituted Pus1‐dependent modification of synthetic mRNAs in vitro. By computational prediction, high‐throughput RNA structure probing, and mutational analysis we have revealed a conserved RNA structure that is necessary and sufficient for mRNA pseudouridylation by Pus1. These results provide insight into how mRNA targets are recognized by pseudouridine synthases, and our experimental approach is applicable to the study of all enzyme‐mRNA interactions.Support or Funding InformationThis work was supported by grants from The American Cancer Society – Robbie Sue Mudd Kidney Cancer Research Scholar Grant (RSG‐13‐396‐01‐RMC) and the National Institutes of Health (GM094303, GM081399) to W.V.G.T.M.C. was supported by the American Cancer Society New England Division (Ellison Foundation Postdoctoral Fellowship).This work was supported in part by the NIH Pre‐Doctoral Training Grant T32GM007287.
Dissecting the Mechanism of H3K36 Methylation in Regulating Pre‐mRNA Splicing
Authors: Leung Calvin , Douglass Stephen , Johnson Tracy 
Published: November 2023

Eukaryotic genes contain introns that are removed during pre‐mRNA splicing by the spliceosome, a large, dynamic RNA‐protein complex composed of five ribonucleoprotein complexes and many associated protein cofactors. Although the detailed mechanism of spliceosome assembly onto pre‐mRNA has been elucidated using elegant biochemistry, in vivo pre‐mRNA splicing is tightly coordinated with gene transcription. Splicing and transcription appear to be coupled not only in time, but also functionally. Spliceosome assembly and the subsequent catalytic steps take place in the context of a dynamic chromatin environment, which suggests that the state of the chromatin may affect splicing. Nonetheless, the precise role of histone modifications in contributing to splicing have remained unclear. Here, we investigate the role of the histone methyltransferase Set2 and its associated chromatin mark, H3K36 methylation, in determining splicing outcomes. Through high‐throughput RNA‐sequencing, we identify discreet classes of lowly expressed transcripts whose splicing is strongly dependent on an intact histone H3K36 methyltransferase complex. Moreover, this same subset of genes shows defective splicing when the H3K36 residue is mutated to prevent its methylation, reinforcing that it is histone methylation per se that is important for splicing these introns. To understand the mechanism by which H3K36 affects splicing, we have analyzed molecular events downstream of H3 methylation, and have identified the NuA4 complex and its chromodomain‐containing protein Eaf3, as a potential mediator of the interaction between the splicing machinery and H3K36 methylation. Here we report our detailed analysis of the mechanism by which this histone modification directly affects spliceosome assembly. Moreover, these studies suggest that subsets of introns may be “tuned” to respond to specific changes in the chromatin environment.Support or Funding InformationNIGMS grant number GM‐085474Ruth L. Kirschstein National Research Service Award GM007185
The Role of the Essential Splicing Factor Prp2 in Ribosome Biogenesis
Authors: Edwards Samantha , Hossain Azad , Johnson Tracy 
Published: November 2023

Ribosome biogenesis is a highly regulated process. Transcription by RNA polymerases I, II, and III is required to generate the RNA and protein components that comprise the ribosome. In addition, the pre‐ribosomal RNA (pre‐rRNA) transcripts must be processed into the mature 18S, 5.8S, 25S, and 5S rRNAs by multiple exo‐ and endonucleolytic cleavages. Ribosomal protein transcripts are also processed via pre‐mRNA splicing. In the yeast Saccharomyces cerevisiae, pre‐mRNA splicing is integral to the regulation of ribosome biogenesis. In fact, out of ~300 intron‐containing genes in yeast, one third are ribosomal protein genes. As a consequence, the production of ribosomal proteins and functional ribosomes is dependent on the process of pre‐mRNA splicing.There have been suggestions in the literature that production of rRNA and ribosomal proteins is coordinated to ensure proper production of functional ribosomes. We have recently uncovered evidence that this coordination occurs by tuning rRNA biogenesis with pre‐mRNA splicing. In a genetic screen to identify genes that regulate the activity of an essential splicing factor Prp2, we uncovered numerous suppressors of the high temperature lethality of the prp2‐1 allele. Surprisingly, among these suppressors are mutations at specific sites within the ribosomal DNA locus. Subsequent analysis has revealed cross talk between the machineries that generate and process rRNA and the ribosomal protein transcripts. Our studies suggest that the splicing and rRNA biogenesis machineries are tuned to maintain the proper stoichiometry of these components. Our results also suggest that this stoichiometry is crucial for proper cellular function. We discuss the possible implications of these findings to understanding ribosome biogenesis and human ribosomopathies.Support or Funding InformationNIGMS grant number GM‐085474Ruth L. Kirschstein National Research Service Award GM007185
Insights into HuR RRM1–2 tandem domains self‐association and mRNA recognition
Authors: Pinheiro Anderson S. , Lixa Carolina , Jendiroba Kleber A. , Lima Luís Maurício T. R. , de Magalhães Mariana T. Q. , Almeida Fabio C. L. 
Published: November 2023

Human antigen R (HuR) employs its mRNA stabilizing activity to function as a major post‐transcriptional regulator of gene expression. This protein plays a central role in the outcome of the inflammatory response as it may stabilize mRNAs of key components of the immune system. HuR comprises three functionally distinct RNA Recognition Motifs: RRM1, RRM2, and RRM3. The two N‐terminal tandem RRM domains can selectively bind AU‐rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly‐A tail of target mRNA. Here, we characterized the structure, dynamics, and binding properties of HuR RRM11‐99 and RRM1–21‐189 tandem domains using a combination of Nuclear Magnetic Resonance and ion mobility‐mass spectrometry. RRM11‐99 adopts a typical RRM fold, composed of two α‐helices and a 4‐stranded β‐sheet (β1α1β2β3α2β4 topology and a β‐hairpin between α2 and β4). 15N relaxation measurements demonstrate that the N‐terminal intrinsically disordered region (residues 1–19) and the RRM1‐RRM2 linker region (residues 95–106) are highly flexible, suggesting that RRM1 and RRM2 domains behave as independent structural modules in solution. NMR titration experiments show that residues on the β‐sheet surface (β1 and β3) and the C‐terminus of RRM11‐99 mediate mRNA binding. An increase in R2/R1 values further confirms the mRNA interaction site, which is compatible with a low μM binding affinity. Both the single RRM11‐99 and the tandem RRM1–21‐189 have the ability to self‐associate, although representing a minor population. Ion mobility‐mass spectrometry identified the presence of oligomers (dimers, trimers and tetramers) with at least three different conformations for the apo forms of RRM11‐99 and RRM1–21‐189. However, only the monomeric species was able to interact with RNA, suggesting that ligand binding shifts the oligomeric/conformational equilibrium of HuR N‐terminal RRM domains. Our results show that the tandem RRM1–2 domains of HuR adopt three distinct conformations in the absence of RNA, suggesting that conformational selection may underlie the mechanism by which HuR recognizes and stabilizes mRNA.Support or Funding InformationThis work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and by a Brazil Initiative Collaboration Grant from Brown University to A.S.P.