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Publications (47)
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. H...
The Warburg effect released lactate promotes cancer progression, but the mechanisms remain unclear. Here, we found lactate activated MAPK pathway through ERK-lactylation to promote cancer progression. Moreover, we identified the GCN5 as the lactyl-transferase for ERK lactylation. Interestingly, activated ERK phosphorylated GCN5 and promoted GCN5 la...
The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20m...
The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20m...
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the ‘writers’ of RNA N⁶-methyladenosine modification, may benefit from poly-(ADP-ribose)-polymerase in...
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitina...
The BRCA1–BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1–10. The BRCA1–BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11–14. The molecular bases for the recruitment to dou...
The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the S. cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic under...
53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully underst...
53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function...
Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation wit...
REV1 is an evolutionarily conserved Translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen (PCNA) is thought to be the trigger for TLS activation. Here we report the solution nuclear magne...
The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechan...
P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the...
PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation reade...
Methyl-lysine (Kme) recognition domains play a central role in epigenetic regulation during cellular differentiation, development, and gene transcription with more than 200 known “reader” domains in the human proteome. We describe our target-class approach to ligand design and discovery for three cancer relevant members of this large family: L3MBTL...
Protein domains of the Royal Family were the first methyllysine binding domains to be discovered. Here, we review what was learned from the structural studies of Royal Family members including chromo, Tudor, MBT, chromo barrel, and PWWP domains. Our main focus is on methyllysine reader domains for which three-dimensional structures are available in...
Individual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses; however, much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 cofactors, including 53BP1 and...
Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward...
p53 is dynamically regulated through various posttranslational modifications (PTMs), which differentially modulate its function and stability. The dimethylated marks p53K370me2 and p53K382me2 are associated with p53 activation or stabilization and both are recognized by the tandem Tudor domain (TTD) of 53BP1, a p53 cofactor. Here we detail the mole...
SynonymsCoherence transfer; Density matrix formalism; Multidimensional NMR; Nuclear magnetic resonance; Product operator formalismSynopsisNuclear magnetic resonance (NMR) is referred to as a nucleus with spin under a magnetic field that will be excited by electromagnetic radiation with specific frequency (Larmor frequency). The resonance frequency...
The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end joining, suggesting that a balance between BRCA1 and 53BP1 regulates DNA double strand-break (DSB) repair mechanism choice. Here we document that acet...
Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium
channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence
and absence of calcium (Ca2+) followed by mass spectroscopic identification of interacting protei...
PHF20 is a multidomain protein and subunit of a lysine acetyltransferase complex that acetylates histone H4 and p53 but whose function is unclear. Using biochemical, biophysical and cellular approaches, we determined that PHF20 is a direct regulator of p53. A Tudor domain in PHF20 recognized p53 dimethylated at Lys370 or Lys382 and a homodimeric fo...
The LOTUS or OST-HTH domain is a recently discovered motif of about 80 amino acids and is found in several germline-specific proteins including the Tudor domain-containing proteins TDRD5 and TDRD7, which are important for germ cell development. The LOTUS domain is an RNA binding domain but its exact function is unknown. Here, we report the 1H, 13C...
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Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome a...
In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the...
Cells have evolved mutagenic bypass mechanisms that prevent stalling of the replication machinery at DNA lesions. This process, translesion DNA synthesis (TLS), involves switching from high-fidelity DNA polymerases to specialized DNA polymerases that replicate through a variety of DNA lesions. In eukaryotes, polymerase switching during TLS is regul...
Bar plots displaying SNF residues that change in peak intensity or chemical shift on titration with U1hpII RNA,poly(U) RNA and self-association. (A) NH signal intensity ratio between free SNF and SNF/U1hpII (1∶7). The residues that display significant concentration-dependent NH peak intensity changes include I18, K20, D39, I40, A42, M48, R49, G50,...
SPR analysis of the interactions between SNF (or SNF RRM1) with U1hpII RNA and poly(U) RNA. SPR analysis was carried out using BIACORE 3000 (Biacore), as described in Materials and Methods. Interactions between U1hpII RNA and SNF RRM1, poly(U) RNA and SNF, poly(U) RNA and SNF RRM1 are shown in A, B and C, respectively. Five different concentrations...
Analytical ultracentrifugation analysis of SNF RRM1 (A) and SNF (B). The protein concentration was about 0.1 mM for SNF RRM1 and 0.09 mM for SNF (2H, 15N, and 13C triple labeled sample), respectively.
(0.32 MB TIF)
The 2D 1H-15N HSQC spectrum of full-length SNF at pH 7.2. Assignments are labeled.
(1.41 MB TIF)
Sequence alignment and surface charge comparison. (A) Structure based sequence alignment of RRM1 and RRM2 in SNF (residues 7–86 and residues 142–216), U1A (residues 10–90 and residues 207–282) and SXL (residues 125–203 and residues 211–291). Conserved residues are shown in red. The secondary structure is displayed at the top. Residues involved in f...
RNA binding assay for SNF and U-120-U (with two U-runs separated by 120 bases), U-120-UC (with one U-run), 2U-13-2U (with two double-U-runs separated by 13 bases) and 2U-13-UC (with one double-U-run) RNA substrates.
(0.63 MB TIF)
Backbone dynamics of full-length SNF. R1, R2, and heteronuclear {1H}-15N NOE values are plotted against residue numbers.
(0.28 MB TIF)
Lysine methylation is an important post-translational modification that affects protein function; for example, the transcriptional activity of the p53 tumor suppressor protein. To facilitate structural characterization of complexes involving proteins and methylated targets by nuclear magnetic resonance spectroscopy, we devised a simple method for p...
Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B'', and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar...
Protein histidine phosphorylation exists widely in vertebrates, and it plays important roles in signal transduction and other cellular functions. However, knowledge about eukaryotic PHPT (protein histidine phosphatase) is still very limited. To date, only one vertebrate PHPT has been discovered, and two crystal structures of hPHPT1 (human PHPT1) ha...
Deacetylation of nucleosomes by the Rpd3S histone deacetylase along the path of transcribing RNA polymerase II regulates access to DNA, contributing to faithful gene transcription. The association of Rpd3S with chromatin requires its Eaf3 subunit, which binds histone H3 methylated at lysine 36 (H3K36). Eaf3 is also part of NuA4 acetyltransferase th...
The Nijmegen breakage syndrome protein Nbs1 is a component of the MRN (Mre11-Rad50-Nbs1) complex, central to the DNA damage response. While Nbs1 is generally believed to encompass a forkhead-associated domain linked to a breast cancer C-terminal (BRCT) domain, to date there is no experimental information on its three-dimensional structure. Through...
Phosphohistidine phosphatase 1 (PHPT1) is the first protein histidine phosphatase identified in vertebrates. The NMR assignments of human PHPT1 are essential for solution structure determination and NMR study of the protein interactions of PHPT1 with its potential substrates.
Protein biotinylation and lipoylation are post-translational modifications, in which biotin or lipoic acid is covalently attached
to specific proteins containing biotin/lipoyl attachment domains. All the currently reported natural proteins containing biotin/lipoyl
attachment domains are multidomain proteins and can only be modified by either biotin...
