Biochemical and Structural Analyses Shed Light on the Mechanisms of RadD DNA Binding and Its ATPase from Escherichia coli.

DNA double-strand breaks (DSBs) are the most perilous and harmful type of DNA damage and can cause tumorigenesis or cell death if left repaired with an error or unrepaired. RadD, a member of the SF2 family, is a recently discovered DNA repair protein involved in the repair of DSBs after radiation or chemical damage. However, the function of RadD in DNA repair remains unclear. Here, we determined the crystal structures of RadD/ATPγS and RadD/ATP complexes and revealed the novel mechanism of RadD binding to DNA and ATP hydrolysis with biochemical data. In the RadD catalytic center, the Gly34 and Gly36 on the P-loop are key residues for ATP binding besides the conserved amino acids Lys37 and Arg343 in the SF2 family. If any of them mutate, then RadD loses ATPase activity. Asp117 polarizes the attacking water molecule, which then starts a nucleophilic reaction toward γ-phosphate, forming the transition state. Lys68 acts as a pocket switch to regulate substrate entry and product release. We revealed that the C-terminal peptide of single-stranded DNA-binding protein (SSB) binds the RadD C-terminal domain (CTD) and promotes the RadD ATPase activity. Our mutagenesis studies confirmed that the residues Arg428 on the zinc finger domain (ZFD) and Lys488 on the CTD of RadD are the key sites for binding branched DNA. Using the Coot software combined with molecular docking, we propose a RadD-binding DNA model for the DNA damage repair process.

Structural basis underlying complex assembly and conformational transition of the type I R-M system.

Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the "open" form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution "closed" structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

Structural basis for DNA recognition and nuclease processing by the Mre11 homologue SbcD in double-strand breaks repair.

The Mre11 complex comprising meiotic recombination 11 (Mre11), Rad50 and Nijmegen breakage syndrome 1 (Nbs1) plays multiple important roles in the sensing, processing and repair of DNA double-strand breaks (DSBs). Here, crystal structures of the Escherichia coli Mre11 homologue SbcD and its Mn2+ complex are reported. Dimerization of SbcD depends on a four-helix bundle consisting of helices α2, α3, α2' and α3' of the two monomers, and the irregular and bent conformation of helices α3 and α3' in the SbcD dimer results in a dimeric arrangement that differs from those of previously reported Mre11 dimers. This finding indicates a distinct selectivity in DNA substrate recognition. The biochemical data combined with the crystal structures revealed that the SbcD monomer exhibits single-stranded DNA (ssDNA) endonuclease activity and double-stranded DNA (dsDNA) exonuclease activity on the addition of a high concentration of Mn2+. For the first time, atomic force microscopy analysis has been used to demonstrate that the SbcD monomer also possesses Mn2+-dependent dsDNA endonuclease activity. Loop ß7-α6 of SbcD is likely to be a molecular switch and plays an important role in the regulation of substrate binding, catalytic reaction and state transitions. Based on structural and mutational analyses, a novel ssDNA-binding model of SbcD is proposed, providing insight into the catalytic mechanism of DSBs repair by the Mre11 complex.

Structural Basis of the Interaction between Human Axin2 and SIAH1 in the Wnt/ß-Catenin Signaling Pathway.

The scaffolding protein Axin is an important regulator of the Wnt signaling pathway, and its dysfunction is closely related to carcinogenesis. Axin could affect the assembly and dissociation of the ß-catenin destruction complex. It can be regulated by phosphorylation, poly-ADP-ribosylation, and ubiquitination. The E3 ubiquitin ligase SIAH1 participates in the Wnt pathway by targeting various components for degradation. SIAH1 is also implicated in the regulation of Axin2 degradation, but the specific mechanism remains unclear. Here, we verified that the Axin2-GSK3 binding domain (GBD) was sufficient for SIAH1 binding by the GST pull-down assay. Our crystal structure of the Axin2/SIAH1 complex at 2.53 Å resolution reveals that one Axin2 molecule binds to one SIAH1 molecule via its GBD. These interactions critically depend on a highly conserved peptide 361EMTPVEPA368 within the Axin2-GBD, which forms a loop and binds to a deep groove formed by ß1, ß2, and ß3 of SIAH1 by the N-terminal hydrophilic amino acids Arg361 and Thr363 and the C-terminal VxP motif. The novel binding mode indicates a promising drug-binding site for regulating Wnt/ß-catenin signaling.

Structure and function of extreme TLS DNA polymerase TTEDbh from Thermoanaerobacter tengcongensis.

Translesion synthesis (TLS) is a kind of DNA repair that maintains the stability of the genome and ensures the normal growth of life in cells under emergencies. Y-family DNA polymerases, as a kind of error-prone DNA polymerase, mainly perform TLS. Previous studies have suggested that the occurrence of tumors is associated with the overexpression of human DNA polymerase of the Y family. And the combination of Y-family DNA polymerase inhibitors is promising for cancer therapy. Here we report the functional and structural characterization of a member of the Y-family DNA polymerases, TTEDbh. We determine TTEDbh is an extreme TLS polymerase that can cross oxidative damage sites, and further identify the amino acids and novel structures that are critical for DNA binding, synthesis, fidelity, and oxidative damage bypass. Moreover, previously unnoticed structural elements with important functions have been discovered and analyzed. These studies provide a more experimental basis for further elucidating the molecular mechanisms of DNA polymerase in the Y family. It could also shed light on the design of drugs to target tumors.

Claudin 18.2 is a potential therapeutic target for zolbetuximab in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is frequently diagnosed and treated in advanced tumor stages with poor prognosis. More effective screening programs and novel therapeutic means are urgently needed. Recent studies have regarded tight junction protein claudin 18.2 (CLDN18.2) as a candidate target for cancer treatment, and zolbetuximab (formerly known as IMAB362) has been developed against CLDN18.2. However, there are few data reported thus far related to the clinicopathological characteristics of CLDN18.2 expression for PDAC. AIM: To investigate the expression of CLDN18.2 in PDAC patients and subsequently propose a new target for the treatment of PDAC. METHODS: The Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Omnibus, and European Genome-phenome Archive databases were first employed to analyze the CLDN18 gene expression in normal pancreatic tissue compared to that in pancreatic cancer tissue. Second, we analyzed the expression of CLDN18.2 in 93 primary PDACs, 86 para-cancer tissues, and 13 normal pancreatic tissues by immunohistochemistry. Immunostained tissues were assessed applying the histoscore. subsequently, they fell into two groups according to the expression state of CLDN18.2. Furthermore, the correlations between CLDN18.2 expression and diverse clinicopathological characteristics, including survival, were investigated. RESULTS: The gene expression of CLDN18 was statistically higher (P < 0.01) in pancreatic tumors than in normal tissues. However, there was no significant correlation between CLDN18 expression and survival in pancreatic cancer patients. CLDN18.2 was expressed in 88 (94.6%) of the reported PDACs. Among these tumors, 50 (56.8%) cases showed strong immunostaining. The para-cancer tissues were positive in 81 (94.2%) cases, among which 32 (39.5%) of cases were characterized for strong staining intensities. Normal pancreatic tissue was identified solely via weak immunostaining. Finally, CLDN18.2 expression significantly correlated with lymph node metastasis, distant metastasis, nerve invasion, stage, and survival of PDAC patients, while there was no correlation between CLDN18.2 expression and localization, tumor size, patient age and sex, nor any other clinicopathological characteristic. CONCLUSION: CLDN18.2 expression is frequently increased in PDAC patients. Thus, it may act as a potential therapeutic target for zolbetuximab in PDAC.

Potent neutralizing RBD-specific antibody cocktail against SARS-CoV-2 and its mutant.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants has posed a serious global public health emergency. Therapeutic interventions or vaccines are urgently needed to treat and prevent the further dissemination of this contagious virus. This study described the identification of neutralizing receptor-binding domain (RBD)-specific antibodies from mice through vaccination with a recombinant SARS-CoV-2 RBD. RBD-targeted monoclonal antibodies (mAbs) with distinct function and epitope recognition were selected to understand SARS-CoV-2 neutralization. High-affinity RBD-specific antibodies exhibited high potency in neutralizing both live and pseudotype SARS-CoV-2 viruses and the SARS-CoV-2 pseudovirus particle containing the spike protein S-RBDV367F mutant (SARS-CoV-2(V367F)). These results demonstrated that these antibodies recognize four distinct groups (I-IV) of epitopes on the RBD and that mAbs targeting group I epitope can be used in combination with mAbs recognizing groups II and/or IV epitope to make mAb cocktails against SARS-CoV-2 and its mutants. Moreover, structural characterization reveals that groups I, III, and IV epitopes are closely located to an RBD hotspot. The identification of RBD-specific antibodies and cocktails may provide an effective therapeutic and prophylactic intervention against SARS-CoV-2 and its isolates.

ATP-dependent conformational change in ABC-ATPase RecF serves as a switch in DNA repair.

RecF is a principal member of the RecF pathway. It interacts with RecO and RecR to initiate homologous recombination by loading RecA recombinases on single-stranded DNA and displacing single-stranded DNA-binding proteins. As an ATP-binding cassette ATPase, RecF exhibits ATP-dependent dimerization and structural homology with Rad50 and SMC proteins. However, the mechanism and action pattern of RecF ATP-dependent dimerization remains unclear. Here, We determined three crystal structures of TTERecF, TTERecF-ATP and TTERecF-ATPɤS from Thermoanaerobacter tengcongensis that reveal a novel ATP-driven RecF dimerization. RecF contains a positively charged tunnel on its dimer interface that is essential to ATP binding. Our structural and biochemical data indicate that the Walker A motif serves as a switch and plays a key role in ATP binding and RecF dimerization. Furthermore, Biolayer interferometry assay results showed that the TTERecF interacted with ATP and formed a dimer, displaying a higher affinity for DNA than that of the TTERecF monomer. Overall, our results provide a solid structural basis for understanding the process of RecF binding with ATP and the functional mechanism of ATP-dependent RecF dimerization.

Crystal structure of the N-terminal domain of human CDC73 and its implications for the hyperparathyroidism-jaw tumor (HPT-JT) syndrome.

CDC73/Parafibromin is a critical component of the Paf1 complex (PAF1C), which is involved in transcriptional elongation and histone modifications. Mutations of the human CDC73/HRPT2 gene are associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome, an autosomal dominant disorder. CDC73/parafibromin was initially recognized as a tumor suppressor by inhibiting cell proliferation via repression of cyclin D1 and c-myc genes. In recent years, it has also shown oncogenic features by activating the canonical Wnt/ß-catenin signal pathway. Here, through limited proteolysis analysis, we demonstrate that the evolutionarily conserved human CDC73 N-terminal 111 residues form a globularly folded domain (hCDC73-NTD). We have determined a crystal structure of hCDC73-NTD at 1.02 Å resolution, which reveals a novel protein fold. CDC73-NTD contains an extended hydrophobic groove on its surface that may be important for its function. Most pathogenic CDC73 missense mutations associated with the HPT-JT syndrome are located in the region encoding CDC73-NTD. Our crystal and biochemical data indicate that most CDC73 missense mutations disrupt the folding of the hydrophobic core of hCDC73-NTD, while others such as the K34Q mutant reduce its thermostability. Overall, our results provide a solid structural basis for understanding the structure and function of CDC73 and its association with the HPT-JT syndrome and other diseases.