The variant form of human xeroderma pigmentosum syndrome (XPV) is caused by a deficiency in DNA polymerase η (Pol η) that enables replication through sunlight-induced pyrimidine dimers. We report high-resolution crystal structures of human Pol η at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol η acts like a molecular splint to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol η orthologs form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. Based on the structures, eight Pol η missense mutations causing XPV can be rationalized as undermining the “molecular splint” or perturbing the active-site alignment. The structures also shed light on the role of Pol η in replicating through D loop and DNA fragile sites.

Native human DNA polymerase η, DNA and dATP were crystallized at pH 6.0 without Mg2+. The polymerization reaction was initiated by exposing crystals to 1 mM Mg2+ at pH 7.0, and stopped at various time points by freezing at 77 K for crystallographic analysis. The substrates and two Mg2+ are aligned for reaction within 40 seconds, but the new bond begins to form at 80 seconds. Structures at reaction times from 80s to 300s reveal a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. In sequence, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo to avoid steric clashes, and the nucleophile and the α-phosphate of dATP approach each other to form the new bond. A third Mg2+ ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.

Major depressive disorder is a serious mental disorder that profoundly affects an individual's quality of life. Although the aetiologies underlying this disorder remain unclear, an increasing attention has been focused on the influence imposed by psychological stress over depression. Despite limited animal models of psychological stress, significant progress has been made as to be explicated in this review to elucidate the physiopathology underlying depression and to treat depressive symptoms. Therefore, we will review classical models along with new methods that will enrich our knowledge of this disorder.

Cisplatin (cis-diamminedichloroplatinum) and related compounds cause DNA damage and are widely used as anticancer agents. Chemoresistance to cisplatin treatment is due in part to translesion synthesis by human DNA polymerase η (hPol η). Here, we report crystal structures of hPol η complexed with intrastrand cisplatin-1,2-cross-linked DNA, representing four consecutive steps in translesion synthesis. In contrast to the generally enlarged and nondiscriminating active site of Y-family polymerases like Dpo4, Pol η is specialized for efficient bypass of UV-cross-linked pyrimidine dimers. Human Pol η differs from the yeast homolog in its binding of DNA template. To incorporate deoxycytidine opposite cisplatin-crosslinked guanines, hPol η undergoes a specific backbone rearrangement to accommodate the larger base dimer and minimizes the DNA distortion around the lesion. Our structural analyses show why Pol η is inefficient at extending primers after cisplatin lesions, which necessitates a second translesion DNA polymerase to complete bypass in vivo. A hydrophobic pocket near the primer-binding site in human Pol η is identified as a potential drug target for inhibiting translesion synthesis and, thereby, reducing chemoresistance.chemotherapy | inhibitor | translesion DNA synthesis | yeast polymerase η

Somatic hypermutation is programmed base substitutions in the variable regions of Ig genes for high-affinity antibody generation. Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T), have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase η (Pol η) is responsible for converting the WA motif to WG by misincorporating dGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol η substituting dGTP for dATP in four sequence contexts, TA, AA, GA, and CA, have been determined and compared. The T:dGTP wobble base pair is stabilized by Gln-38 and Arg-61, two uniquely conserved residues among Pol η. Weak base paring of the W (T:A or A:T) at the primer end and their distinct interactions with Pol η lead to misincorporation of G in the WA motif. Between two WA motifs, our kinetic and structural data indicate that A-to-G mutation occurs more readily in the TA context than AA. Finally, Pol η can extend the T:G mispair efficiently to complete the mutagenesis.

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H2O2, OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H2O2, but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H2O2, but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR.

SummaryTwo-component systems are predominant signal transduction pathways in prokaryotes, and also exist in many archaea as well as some eukaryotes. A typical TCS consists of a histidine kinase and a cognate response regulator. In this study, a novel gene encoding a response regulator (we designate it drRRA) is identified to be essential for the extreme radioresistance of Deinococcus radiodurans. DrRRA null mutant (we designate it MR) is sensitive to gamma-radiation compared with the wild-type strain. Transcriptional assays show that numerous genes are changed in their transcriptional levels in MR at exponential growth phase under normal or gammaradiation condition. Most of them are related to stress response and DNA repair. Antioxidant activity assays exhibit that both superoxide dismutases and catalases are decreased in the mutant, whereas Western blotting assays show that RecA and PprA are also reduced in MR, verifying the microarray and quantitative real-time PCR data. Furthermore, pulsed-field gel electrophoresis assay demonstrates that deletion of drRRA results in the delay of genome restitution. These data support the hypothesis that DrRRA contributes to the extreme radioresistance of D. radiodurans through its regulatory role in multiple pathways such as antioxidation and DNA repair pathways.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a membrane glycoprotein expressed by activated effector T cells (Teffs) and participates in the repression of T cell proliferation, cell cycle progression and cytokine production. Currently, antibodies targeting CTLA-4, ipilimumab and tremelimumab are widely used as a therapeutic approach in a variety of human malignancies. However, their detailed mechanism remains unclear. Therefore, in this review, we focused specifically on recent findings concerning the role of CTLA-4 in immune response and also discussed clinical studies of targeting CTLA-4, alone or in combination with other therapies for the treatment of cancers. CTLA-4 blockade is used as a therapeutic approach for the treatment of cancer through competing with CD28-positive costimulation for binding to their shared B7 ligands or exhibiting direct inhibitory effect on signaling molecules in the cytoplasmic tail. At present, antibodies for targeting CTLA-4 or in combination with other therapies significantly reinforced the anti-tumor effect and improved the prognosis of malignant disease. In addition, severe adverse events of targeting CTLA-4 therapy could be a challenge for the development of this therapeutic strategy. This review may provide some new insights for clinical studies of targeting CTLA-4.