Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

Fabrication and characterization of spearmint oil loaded nanoemulsions as cytotoxic agents against oral cancer cell

Spearmint oil (SMO), a commonly used essential oil for oral care products, possesses various interesting functions, especially for anticancer property. However, the application of SMO for cancer treatment is limited due to water insoluble. In the present study, nanoemulsions, which have been widely accepted as dosage forms for poorly water-soluble drugs, were selected as candidate carriers for SMO to inhibit oral cancer cell. The nanoemulsions were fabricated using phase inversion temperature method. The factors affecting formation and properties of nanoemulsions including type and amount of surfactants, oil loading and ratio of SMO to virgin coconut oil (VCO) were investigated. Among the surfactants used, the nanoemulsions containing polyoxyethylene castor oil derivatives (Kolliphor®EL; PCO35, Cremophor®RH40; PCO40, Eumulgin®CO60; PCO60) and polyoxyethylene sorbitan fatty acid esters (PSF80) showed 100% creaming after temperature cycling test indicating excellent physical stability while those containing PCO40 demonstrated more transparency and better physical stability. With an increasing amount of PCO40, the droplet size tended to decrease and was in the nano-size range (<1000 nm) after increasing to more than 5% (w/w). SMO-VCO loading also influenced on the droplet size. At 5% (w/w) PCO40, the maximum SMO-VCO loading of 25% (w/w) to attain nanoemulsions was observed. Moreover, the composition of oils had an impact on size of emulsions. The transparent nanoemulsions were only prepared in the range of SMO-VCO from 40:60 to 80:20, suggesting the optimum ratio of SMO to surfactant and the composition of oils were the critical factors for formation of nanoemulsions. NMR study disclosed that the interaction between PCO40 with both VCO and SMO should be a possible stabilization mechanism. Furthermore, the SMO-VCO nanoemulsions exhibited significant cytotoxic effect against oral carcinoma (KON) cell line using MTT assay. The finding, therefore, revealed the good feasibility of SMO-VCO nanoemulsions as novel carriers for treating of oral cancer. Keywords: Nanoemulsions, Spearmint oil, Coconut oil, Surfactants, Anticancer, NM

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation

CV1720 cells show impaired ATR–dependent DNA damage responses.

<p>A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm⁻² UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm⁻²) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.</p

Clinical features of ATR/ATRIP–deficient patients.

<p>NR; not recorded. NA; not assessed.</p

Identification of mutational changes in <i>ATRIP</i> in CV1720.

<p>A) Upper panel shows primer pairs used to distinguish cDNA products encompassing or lacking exon 2. Lower panel shows RT-PCR products obtained using the primers shown in the upper panel. RT-PCR from patient CV1720 generated a smeared product with a defined band of 458 bp, as observed in WT cells, and a weaker band of 325 bp. The latter band was not detected using cDNA from WT cells (MRC5). A similar single 458 bp band was obtained using the same primers with cDNA derived from a distinct wild type cell line (GM2188; data not shown). (B) Sequencing of the RT-PCR products derived from WT (MRC5) and patient (CV1720) cells. A double sequence pattern at the exon 2–3 boundary is observed using patient CV1720 cDNA. C) Selective quantitative amplification of the WT or 2278C>T <i>ATRIP</i> alleles. Primers located in <i>ATRIP</i> exon 12 and 13 were designed to selectively amplify the WT (c.2278C) (P1 and P3C) versus the mutated (c.2278C>T) (P2 and P3C) alleles. The WT PCR product is shown in blue and the c.2278C>T PCR product in red. The exon 12 mutated allele is only observed in the patient and mother cDNA whilst the WT allele is observed in the patient, mother and father cDNA although the level is reduced in the patient and mother. D) qRT-PCR analysis of <i>ATRIP</i> splicing variants from patient CV1720 and parental cells. qRT-PCR analysis of the level of the normally spliced (encompassing exons 1-2-3) and the aberrantly spliced (Δexon2) <i>ATRIP</i> cDNA in the patient and parent cells. PCR primers were designed at the exon2-exon3 or exon1-exon3 boundaries to selectively amplify the splicing variants. Transcripts from <i>HPRT1</i> were used as a quantification control. The correctly spliced transcript from the paternal allele of the patient (wild type c.2278C, blue fraction in the cumulative bar labelled, ‘patient’, at the left panel) was estimated to be ∼25% of the normal level. (E) The mis-spliced paternal allele is subject to nonsense mediated mRNA decay (NMD). Cycleave-qPCR confirmed that the <i>ATRIP</i> c.2278C>T mutant allele was expressed exclusively in the patient and the mother. The <i>ATRIP</i> exon12-13 fragment was amplified with PCR primers P7/P8 as shown in the figure. A set of fluorescent probes were used to distinguish the WT versus c.2278C>T allele (probe1 and probe2, respectively). In the patient, the paternal mRNA transcript level (emerald lines) is low because of NMD (top left). Puromycin treatment eliminated the NMD and the paternal transcript level returned to the normal level. In all panels WT represented MRC5, patient was CV1720 and parents were as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen-1002945-g001" target="_blank">Figure 1A</a>.</p

MGS and Seckel syndrome patient phenotypes.

*<p>standard deviations from the age-related normal population mean, NA = not assessed.</p><p>MGS data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell2" target="_blank">[14]</a><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Guernsey1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-deMunnik1" target="_blank">[34]</a>.</p

Patients 27-4BI and 19-8BI have reduced ATR and ATRIP expression and mutations in ATR.

<p>A) Photographs of patient included with informed consent of parent. B) Cell extracts (50 µg) from LBLs derived from WT (IM257), patient 27-4BI or patient 19-8BI were immunoblotted using the indicated antibodies. Reduced expression of ATR was observed in both patients. 27-4BI also had reduced ATRIP expression. C) Structure of ATR showing the site of the mutations identified and the UME domain. D) The UME domain is conserved between species and the methionine residue within this domain is conserved in yeast.</p