DNA Repair

Interstrand crosslinks are cytotoxic lesions that inhibit essential processes including replication and transcription. Replication fork reversal occurs in response to interstrand crosslink inducing drug, MMC, but how replication fork reversal promotes repair of interstrand crosslinks is poorly understood. Here, we investigated the role of the RAD54L translocase in interstrand crosslink repair. We found RAD54L is required to promote nascent DNA degradation in FANCD2 and FANCA-depleted cells consistent with a previous study indicating RAD54L promotes replication fork reversal. We further show RAD54L activity is required for formation of radial chromosomes in FANCD2-deficient cells suggesting fork reversal may be required to generate the intermediate undergoing aberrant fusion in FANC-deficient cells. Finally, we demonstrate FANCD2 foci accumulate and DSBs persist in RAD54L-deficient cells indicating RAD54L is required for efficient repair of DSBs. Together, our results indicate RAD54L plays multiple roles in efficient processing and repair of interstrand crosslinks.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies. Immune dysregulation is believed to play an important role in ATC. Here, we aimed to characterize the systemic inflammation and the function of circulating immune cells of patients with ATC. First, we retrospectively assessed biochemical parameters of patients with ATC and observed that high systemic inflammation correlated with worse survival. Next, we prospectively investigated the inflammatory proteome, single-cell peripheral blood mononuclear cell transcriptome and epigenetic changes. Circulating concentrations of proinflammatory cytokines were increased in ATC patients. This proinflammatory profile was apparent at the level of gene transcription and chromatin accessibility, especially in monocytes. These findings were substantiated by an increased capacity of peripheral blood mononuclear cells of ATC patients to produce IL-6, IL-8 and lactate. As IL-6 is known to promote tumor cell survival, we assessed its capacity to influence ATC cell proliferation. Blocking IL-6/gp130/Jak/STAT3 pathway inhibited proliferation of ATC cell lines in vitro. In conclusion, these findings show that ATC is characterized by inappropriate systemic inflammation and epigenetic and transcriptional reprogramming of circulating monocytes. Proinflammatory cytokines released by monocytes support survival and proliferation of ATC tumor cells, suggesting a therapeutic potential of targeting this pathway in ATC patients.

The repair of photo-induced DNA lesions through nucleotide excision repair machinery is still the source of important questions. It has been observed that the repair rate of the different cyclobutane pyrimidine dimers, i.e. the photoproducts induced by dimerization of two {pi}-stacked pyrimidines (T<>T, T<>C, C<>T, C<>C), depends on the nucleobases involved in the lesion. TT derivatives (T<>T) are removed more slowly than those containing cytosine, especially in 5. Using all-atom molecular dynamics simulations and free-energy calculations, we demonstrate that the variation of the repair rate observed in human skin and in cultured cutaneous cell is associated to the recognition of the four lesions by the DDB2 protein moiety, and more specifically by the differential structural deformation induced on the complementary strand. Indeed, while C<>C and C<>T induce a larger deviation on the groove parameters, T<>T and T<>C, instead, affect DNA structure to a lesser extent. less affected. These effects then hamper differentially the downstream recruitment of the repair complexes. The observed DNA deformation correlates with the experimental repair rate and provides a structural rationale for the different repair rates of CPD by nucleotide excision repair machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/724087v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@cf6b6dorg.highwire.dtl.DTLVardef@195e35forg.highwire.dtl.DTLVardef@1829296org.highwire.dtl.DTLVardef@165baba_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

Type III restriction-modification (RM) enzymes are prominent bacterial defense against bacteriophage and invading foreign DNA that also modulate the hosts epigenetic landscape. Genome analysis of the host-adapted Mycoplasma bovis PG45 that has a very small genome revealed a Type III RM locus comprising one res and three mod genes. We characterized Mbo45V, a representative enzyme encoded by this locus. The enzyme forms a heterotrimeric complex consisting of two Mod subunits and one Res subunit. Mbo45V recognizes the asymmetric sequence 5'-YAATC-3' (Y = T/C) and cleaves DNA having at least two head-to-head oriented sites [~]26-28 bp away from the recognition site. Methylation of the second adenine of the target site using cofactor S-adenosylmethionine (SAM) protects DNA from restriction, while the SAM analogue sinefungin enhances DNA binding and cleavage. Kinetic studies reveal that Mbo45V exhibits relatively weak DNA binding affinity and an unusually high Km for SAM, indicating low cofactor affinity compared to prototypical enzymes such as EcoP15I. ATPase activity is strongly stimulated by cognate DNA and is inhibited upon methylation of the substrate, suggesting a regulatory interplay between methylation and restriction functions. Comparative analysis indicates that, although Mbo45V shares core mechanistic features with prototypes from Escherichia coli, its kinetic parameters are distinct. These differences likely reflect adaptation to the stable intracellular environment of M. bovis, in contrast to the fluctuating conditions encountered by the enteric bacteria.

DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are E. coli UvrD, Rep, B. stearothermophilus PcrA and M. tuberculosis UvrD1. SF1A monomers are processive 3 to 5 single stranded DNA translocases, but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.

The cellular response to DNA replication stress (DRS) provoked by anticancer drugs involves activation of the G2/M checkpoint (which promotes transient cell cycle arrest at G2 phase) and DNA repair, followed by induction of apoptosis or senescence. Here, we activated the p53-p21 pathway and ATR using DRS-inducing drugs, and found that that the transition to senescence depends on the duration of the G2 phase. Shortening of G2 duration by G2/M checkpoint inhibitors led not only to a switch in cell fate from senescence to mitotic entry, but also to effective cell death through carry-over of chromosomal aberrations (generated by DRS-inducing drugs) into mitosis and subsequent mitotic progression. Such enhanced cell death was also observed in p53 deficient cells, which do not normally undergo senescence. Thus, we propose that temporal regulation of G2 phase is an approach to enhancing the effects of DRS-inducing drugs in a manner that is independent of p53 status.

RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/723793v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13899a1org.highwire.dtl.DTLVardef@ee1aadorg.highwire.dtl.DTLVardef@1991e1org.highwire.dtl.DTLVardef@b877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

The anticancer efficacy of radiotherapy (RT) is limited by acquired radioresistance (RR). Here, we aim to characterize prolonged responses of breast carcinoma cells to hypofractionated irradiation (hFI). To this end, murine mammary 4T1 tumor cells (4T1WT) were subjected to a clinically oriented hFI protocol (56 Gy cumulative dose) to select radioresistant cells in vitro (4T1RS). Furthermore, hFI of subcutaneously growing 4T1CTR tumors (hFI; 24 Gy cumulative dose) was performed to radioselected 4T1IR cells in vivo. Following single irradiation in vitro, radioselected 4T1RS cells revealed increased proliferation, attenuated G2/M arrest and reduced apoptosis as compared to parental 4T1WT cells. Moreover, 4T1RS cells showed increased expression of DNA-damage response (DDR)-related proteins (pKAP1, pCHK2, {gamma}H2AX) and improved DSB repair efficiency as demonstrated by nuclear {gamma}H2AX foci analyses. The mRNA expression of factors regulating cell cycle progression, DDR, apoptosis and oxidative stress was substantially different between both cell variants in vitro. Ten days after hFI of in vivo growing tumors, residual DNA damage and apoptosis were increased in the radioselected 4T1IR tumors, whereas proliferation was reduced as compared to non-irradiated 4T1CTR control tumors. Both irradiated and non-irradiated tumors revealed complex differences in the mRNA expression profile of susceptibility- and metastasis related genes, including GADD45a, DUSP1, CDKN1a and NQO1 as well as CD44 and Rho-related factors, respectively. Moreover, hFI stimulated the infiltration of MPO-positive immune cells into tumor tissue while the presence of CD3-positive cells was reduced in the tumor area. In addition, hFI in vivo resulted in a dysregulated mRNA expression of various immune cell markers, Rho-regulatory factors, tissue remodeling molecules and cell adhesion factors. Summarizing, we identified long-lasting adaptive changes following hFI in vitro and in vivo that are associated with DNA replication, DNA repair, senescence and apoptosis as well as immune cell infiltration and tissue remodeling.

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ionizing radiation induces molecular responses that may be used to estimate exposure when physical dosimeters are unavailable. Here we present two large-scale proteomics datasets generated from mouse dorsal skin punch samples collected following controlled X-ray exposures spanning multiple doses, dose rates, and post-exposure time points. Experiment 1 comprised 96 samples (including 16 reference samples) collected 6 days after exposure to 0-75 cGy delivered at either 30 or 300 cGy/min. Experiment 2 comprised 936 samples (including 236 reference samples) exposed to 0-100 cGy at either 3 or 28 cGy/min dose rates and harvested between 7 and 150 days post-exposure. All samples were processed using a standardized workflow involving automated bead-based digestion and data-independent acquisition mass spectrometry. The datasets include multiple pooled reference sample types, process controls, and system suitability standards ensuring high quality data. All data presented are available via ProteomeXchange at several levels of processing, from raw files through normalized peptide- and protein-level abundance matrices suitable for biomarker discovery and machine learning applications. This dataset will facilitate generation of new insights into the biological changes and molecular signatures resulting from X-ray exposure in mice and may also help inform future studies in humans.

PurposeGlasdegib is a Sonic Hedgehog (SHH) pathway inhibitor used for treating newly diagnosed acute myeloid leukemia in elders or patients unfit for intensive chemotherapy. This study sought to demonstrate growth inhibition and increased apoptosis of B-cell acute lymphoblastic leukemia (B-ALL) in vitro under glasdegib, alone and combined with inotuzumab, using a novel co-culture system and validated chemosensitivity testing model to determine whether glasdegib with and without inotuzumab may represent a promising treatment strategy in B-ALL. MethodsSeven blood and marrow samples from B-ALL patients were co-cultured with HS-5 stromal cells in a co-culturing system designed to mimic the tumor microenvironment to maintain B-ALL cell viability for chemosensitivity testing under glasdegib and inotuzumab. ResultsCo-culturing improved B-ALL viability from four to nine days. Dosage-dependent responses to glasdegib were consistent among B-ALL samples on day four based on culture viability, and varied based on expressions of SSH genes GLI1, GLI3, SMO, and PTCH1. Combination with inotuzumab had varied effects on treatment response. ConclusionCo-culturing B-ALL cells with HS-5 stromal cells improves B-ALL growth and viability. Glasdegib with and without inotuzumab treatments impact the viability of co-cultured B-ALL cells by day four. SHH gene expressions suggest different B-ALL patients may be sensitive or resistant to glasdegib and inotuzumab.

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Organismal survival depends on coordinated responses to oxidative stress and DNA damage. Using Caenorhabditis elegans, we investigate mul-1, a robust transcriptional target of ionizing radiation and reactive oxygen species. Although annotated as a mucin, MUL-1 is a small ShKT domain-containing protein belonging to an invertebrate expanded family of cysteine-rich proteins. mul-1 is selectively induced by oxidative stress, including IR, hydrogen peroxide (H2O2), Pseudomonas aeruginosa infection, or loss of the peroxiredoxin PRDX-2, via the p38 MAPK-ATF-7 pathway in intestinal cells. Loss of mul-1 and its paralogs increases ROS accumulation, oxidative stress sensitivity, and CEP-1/p53 dependent germ cell apoptosis. Combined deletion of mul-1 paralogs causes constitutive apoptosis, reduced fecundity, and compensatory activation of DAF-16/Foxo and SKN-1/Nrf2 stress response pathways. Together with genetic analysis of SYSM-1, these findings suggest MUL-1-like ShKT proteins buffer oxidative stress.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

Targeted protein degradation using conditional degron tag (CDT) technology is a powerful method for rapidly degrading a protein of interest (POI) upon the addition of a degrader drug. A prerequisite for the temporally controlled degradation of an endogenous POI is the generation of homozygous knock-in cells with the degron tag integrated at either the N- or C-terminus of their gene loci. However, obtaining those homozygous knock-in cells often requires selecting many single-cell clones, as human cells typically exhibit low homology-directed repair (HDR) activities. Additionally, tagging a degron to an endogenous protein may inadvertently reduce protein expression, potentially affecting protein function even before the drug is administered. Here, we develop a method for generating degron-tagged knock-in cells that allows us to skip the laborious single-cell cloning. This method arose from our observation that most knock-in cells carry the degron tag only in one allele (heterozygous), while the other allele typically harbors a frameshift insertion/deletion. This observation allowed us to bypass the need for single-cell cloning. We validated our method by knocking in degron tags at the N-terminus of cytoplasmic dynein1 subunits or Adaptor Protein 2 (AP2) subunit. Our experiments confirmed the rapid degradation of these proteins and their functional inhibition in bulk cell populations. Additionally, to mitigate the reduced expression often associated with the degron tagging, we established a method to control expression levels by inserting a mini-promoter immediately upstream of the knock-in cassette. Our method simplifies the workflow for degron tag knock-ins and enhances the versatility of these valuable technologies.