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1.
Nat Commun ; 15(1): 2064, 2024 Mar 07.
Article in English | MEDLINE | ID: mdl-38453899

ABSTRACT

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.


Subject(s)
Serine Endopeptidases , Serine Proteases , Dimerization , Serine Endopeptidases/metabolism , Proteolysis , DNA Replication , Serine
2.
Nucleic Acids Res ; 51(5): 2117-2136, 2023 03 21.
Article in English | MEDLINE | ID: mdl-36715322

ABSTRACT

The conserved complex of the Rad6 E2 ubiquitin-conjugating enzyme and the Bre1 E3 ubiquitin ligase catalyzes histone H2B monoubiquitination (H2Bub1), which regulates chromatin dynamics during transcription and other nuclear processes. Here, we report a crystal structure of Rad6 and the non-RING domain N-terminal region of Bre1, which shows an asymmetric homodimer of Bre1 contacting a conserved loop on the Rad6 'backside'. This contact is distant from the Rad6 catalytic site and is the location of mutations that impair telomeric silencing in yeast. Mutational analyses validated the importance of this contact for the Rad6-Bre1 interaction, chromatin-binding dynamics, H2Bub1 formation and gene expression. Moreover, the non-RING N-terminal region of Bre1 is sufficient to confer nucleosome binding ability to Rad6 in vitro. Interestingly, Rad6 P43L protein, an interaction interface mutant and equivalent to a cancer mutation in the human homolog, bound Bre1 5-fold more tightly than native Rad6 in vitro, but showed reduced chromatin association of Bre1 and reduced levels of H2Bub1 in vivo. These surprising observations imply conformational transitions of the Rad6-Bre1 complex during its chromatin-associated functional cycle, and reveal the differential effects of specific disease-relevant mutations on the chromatin-bound and unbound states. Overall, our study provides structural insights into Rad6-Bre1 interaction through a novel interface that is important for their biochemical and biological responses.


Subject(s)
Histones , Saccharomyces cerevisiae Proteins , Ubiquitin-Conjugating Enzymes , Humans , Chromatin/genetics , Chromatin/metabolism , Histones/genetics , Histones/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism
3.
Nat Commun ; 11(1): 1318, 2020 03 12.
Article in English | MEDLINE | ID: mdl-32165630

ABSTRACT

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.


Subject(s)
DNA Replication , Receptors, Virus/metabolism , Trypsin/chemistry , Camptothecin/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , DNA Damage , DNA Topoisomerases, Type I/metabolism , DNA, Single-Stranded/metabolism , Humans , Mutation/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding/drug effects , Protein Domains , Receptors, Virus/chemistry , Receptors, Virus/genetics
4.
Nanomedicine ; 14(7): 2465-2474, 2018 10.
Article in English | MEDLINE | ID: mdl-28554596

ABSTRACT

Two-dimensional (2D) nanomaterials are an emerging class of materials with unique physical and chemical properties due to their high surface area and disc-like shape. Recently, these 2D nanomaterials have been investigated for a range of biomedical applications including tissue engineering, therapeutic delivery and bioimaging, due to their ability to physically reinforce polymeric networks. Here, we present a facile fabrication of a gradient scaffold with two natural polymers (gelatin methacryloyl (GelMA) and methacrylated kappa carrageenan (MκCA)) reinforced with 2D nanosilicates to mimic the native tissue interface. The addition of nanosilicates results in shear-thinning characteristics of prepolymer solution and increases the mechanical stiffness of crosslinked gradient structure. A gradient in mechanical properties, microstructures and cell adhesion characteristics was obtained using a microengineered flow channel. The gradient structure can be used to understand cell-matrix interactions and to design gradient scaffolds for mimicking tissue interfaces.


Subject(s)
Cell Adhesion , Hydrogels/chemistry , Mesenchymal Stem Cells/cytology , Nanocomposites/chemistry , Polymers/chemistry , Tissue Engineering , Tissue Scaffolds , Cells, Cultured , Humans , Rheology , Silicates/chemistry
5.
Elife ; 62017 08 16.
Article in English | MEDLINE | ID: mdl-28826505

ABSTRACT

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.


Subject(s)
Histone Chaperones/chemistry , Histone Chaperones/metabolism , Protein Processing, Post-Translational , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/metabolism , Crystallography, X-Ray , Models, Molecular , Phosphorylation , Protein Binding , Protein Conformation , Transcription, Genetic
6.
Nat Commun ; 7: 11949, 2016 06 21.
Article in English | MEDLINE | ID: mdl-27325136

ABSTRACT

Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation-demethylation together control chromatin dynamics during various facets of transcriptional regulation.


Subject(s)
Genome, Fungal , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Chromatin Assembly and Disassembly , Gene Expression Regulation, Fungal , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Methylation , Models, Genetic , Multigene Family , Nucleosomes/chemistry , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic
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