DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

MiR-210在缺氧中调控机制的研究进展

MicroRNA(miRNA)是一类包含21-25个核苷酸的单链非编码小RNA.研究表明,miR-210可直接抑制线粒体内铁硫蛋白(Fe-S)的支架蛋白ISCUl/2的表达从而抑制线粒体代谢;miR-210对DNA损伤修复基因-RAD家族有抑制作用,减弱了DNA修复能力;miR-210可以通过调节E2F3、纤维母细胞生长因子受体1(FGFRL1)、同源域基因A1(HOXA1)等阻止细胞增殖,调控细胞周期;缺氧刺激可上调miR-210表达,这对促进血管再生有重要作用.miR-210的表达受缺氧诱导,调控缺氧反应相关基因的表达,提示miR-210有可能成为诊治包括缺血性损伤、肿瘤在内等多种疾病的新靶点.     

Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.     

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3′-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (“long-patch”) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5′-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15–30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60–70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5′-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.     

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.     

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior-posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.     

Homozygous deficiency of ubiquitin-ligase ring-finger protein RNF168 mimics the radiosensitivity syndrome of ataxia-telangiectasia

Maintaining genomic integrity is critical to avoid life-threatening disorders, such as premature aging, neurodegeneration and cancer. A multiprotein cascade operates at sites of DNA double-strand breaks (DSBs) to recognize, signal and repair damage. RNF168 (ring-finger nuclear factor) contributes to this emerging pathway of several E3 ubiquitin ligases that perform sequential ubiquitylations on damaged chromosomes, chromatin modifications essential for aggregation of repair complexes at the DSB sites. Here, we report the clinical and cellular phenotypes associated with a newly identified homozygous nonsense mutation in the RNF168 gene of a patient with a syndrome mimicking ataxia-telangiectasia. The mutation eliminated both of RNF168's ubiquitin-binding motifs, thus blocking progression of the ubiquitylation cascade and retention of repair proteins including tumor suppressors 53BP1 and BRCA1 at DSB sites, consistent with the observed defective DNA damage checkpoints/repair and pronounced radiosensitivity. Rapid screening for RNF168 pathway deficiency was achieved by scoring patients' lymphoblastoid cells for irradiation-induced nuclear foci containing 53BP1, a robust assay we propose for future diagnostic applications. The formation of radiation-induced DSB repair foci was rescued by ectopic expression of wild-type RNF168 in patient's cells, further causally linking the RNF168 mutation with the pathology. Clinically, this novel syndrome featured ataxia, telangiectasia, elevated alphafetoprotein, immunodeficiency, microcephaly and pulmonary failure and has implications for the differential diagnosis of autosomal recessive ataxias.     

NITROGEN LIMITATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) LEADS TO INCREASED SUSCEPTIBILITY TO DAMAGE BY ULTRAVIOLET‐B RADIATION BUT ALSO INCREASED REPAIR CAPACITY1

Chl fluorescence was used to measure the photosynthetic capacity of the green alga Dunaliella tertiolecta in order to investigate interactions between susceptibility to acute UV‐B radiation (UVBR, 280–320 nm) exposure and decreased nitrogen availability. Under UVBR exposure the decline in the fluorescence parameters F_v/F_m (the maximum effective quantum yield Φ_PSIIe‐max) and F_v′/F_m′ (the operational quantum yield of PSII, Φ_PSIIe) were enhanced with higher UVBR fluxes, with the data well described by the Kok model, inferring that a dynamic balance existed between damage and repair with the repair proportional to the pool size of inactivated targets. When UVBR exposure was coupled with nitrogen limitation, the inhibition of photosynthesis was intensified. Under the more severely N‐limited conditions, the damage rate increased. Unexpectedly, repair rates were also stimulated under N‐limited conditions, although this was insufficient to counteract the increase in damage, so the overall effect of N limitation was an enhancement of UVBR‐induced inhibition of photosynthesis.