Biology subjects

Min, J. H.

Publications and source records attributed to Min, J. H..

7 recordsLinked to original sources

bioRxiv2025

Human placental stem cells induce a novel multiple myeloid cell-driven immunosuppressive program that ameliorates proinflammatory CNS pathology

Despite a growing interest in Amniotic Epithelial Cell (AEC)-based therapies, the immune responses triggered by AEC transplantation in vivo remain poorly characterized. In particular, how direct exposure to AECs within the central nervous system (CNS) shapes the local immune environment is currently unknown. Herein we describe a novel CNS- specific immunoregulatory pathway induced by intracisternal delivery of human AECs. Local immune responses induced by AECs in the brain led to recruitment of immunosuppressive Arginase 1+ (ARG1+) macrophages and a novel population of myeloid-derived suppressor cells with eosinophilic characteristics, which we term Eo- MDSCs. We further demonstrate that Eo-MDSCs produce Maresin 2 (MaR2), a specialized pro-resolving mediator (SPM) involved in the resolution of inflammation. In a mouse model of Multiple Sclerosis (MS), treatment of established disease with AECs induced immunological responses that resulted in reduced numbers of pathogenic macrophages and T helper (TH)17 cells, increased anti-inflammatory T cell subsets, and enhanced myelin phagocytosis, all of which led to functional recovery. These findings suggest that AEC therapy has the potential to target CNS-intrinsic inflammatory processes in MS, providing a strong rationale for translation into the clinic.

neuroscience↗

bioRxiv2024

Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair complex

Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. We examined DNA dynamics related to damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Using laser temperature-jump (T-jump), we measured the dynamics of DNA containing 3 base-pair mismatches recognized specifically by Rad4 in vitro. The T-jump kinetics traces measured using a cytosine-analog FRET pair, together with rigorous comparison with equilibrium measurements, enabled conformational dynamics to be revealed beyond the T-jump observation window of [~]20 {micro}s - 50 ms. AT-rich nonspecific sites (matched or mismatched) exhibited dynamics primarily within the T-jump window, albeit with some amplitude in "missing" fast (< 20 {micro}s) kinetics. The fast-kinetics amplitudes increased dramatically for specific sites, which were further distinguished by additional missing amplitude in slow (> 50 ms) kinetics at elevated temperatures. We posit that the rapid ({micro}s-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that likely govern how Rad4 senses DNA damage.

biophysics↗

bioRxiv2021

Structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in Nucleotide Excision Repair

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit core TFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 [A] resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3 and 5 side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 would extend the DNA opening at the lesion and deliver the damaged strand to Rad3 (XPD) in an unwound form suitable for subsequent lesion scanning and verification.

biophysics↗

bioRxiv2020

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively opening these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a kinetic gating mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how opening is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed open undamaged DNA in solution and that such opening can largely occur without one or the other of the {beta}-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound open DNA adopts multiple conformations in solution notwithstanding the DNAs original structure or the {beta}-hairpins. Molecular dynamics simulations reveal compensatory roles of the {beta}-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA opening of undamaged sites and the dynamic nature of open DNA may shed light on how the protein functions within and beyond NER in cells.

biochemistry↗

bioRxiv2020

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes with high spatial and temporal resolutions is difficult as it requires ways to rapidly trigger the assembly/disassembly as well as ways to monitor the structural changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, thereby their activities. Here we report that photoswitchable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically binds to diverse helix-destabilizing/distorting lesions including bulky organic adducts and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding renders it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA can be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/313114v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f31fc7org.highwire.dtl.DTLVardef@ab512dorg.highwire.dtl.DTLVardef@15e8493org.highwire.dtl.DTLVardef@51f58d_HPS_FORMAT_FIGEXP M_FIG Graphical Summary This work shows that a photolabile 6-nitropiperonyloxymethyl (NPOM)-modified DNA is specifically recognized by the Rad4/XPC damage sensor protein complex that initiates the nucleotide excision repair pathway; light-induced cleavage of NPOM abolishes the specific binding to Rad4/XPC. C_FIG

biochemistry↗

bioRxiv2020

The DNA Damage-Sensing NER Repair Factor XPC-RAD23B Does Not Recognize Bulky DNA Lesions with a Missing Nucleotide Opposite the Lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were [~] 2.5 - 3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 {beta}-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

biochemistry↗

bioRxiv2020

Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

Rad4/XPC is a key DNA damage sensor for nucleotide excision repair (NER) in eukaryotes. Rad4/XPC recognizes diverse bulky lesions by flipping out two lesion-containing nucleotide pairs and inserting a {beta}-hairpin from the BHD3 domain ({beta}-hairpin3) into the DNA duplex. We have previously observed that Rad4 can form the same open structure when covalently tethered to a normal DNA sequence containing consecutive C/Gs (CCC/GGG) and that a similar open-like structure can be formed even when the {beta}-hairpin3 is lacking. Here, we report a crystal structure of the {Delta}{beta}-hairpin3 mutant tethered to a sequence containing alternating C/Gs (CGC/GCG). In contrast to the previous structures, Rad4 bound to CGC/GCG in a 180{degrees}-reversed manner, capping the end of the duplex without flipping out the nucleotides. MD simulations showed that CGC/GCG was inherently less openable than CCC/GGG and that Rad4 failed to engage with its minor groove, a hallmark of productive binding towards opening. These results reveal that DNA sequences significantly influence the thermodynamic barrier for DNA opening by Rad4, which may render certain DNA structures/sequences resistant to opening despite a long residence time of Rad4. The reverse- mode may indicate unproductive binding for NER whereas the DNA end-binding may hint at Rad4/XPCs functions beyond NER.

biochemistry↗