The role of mammalian NEIL1 protein in the repair of 8-oxo-7,8-dihydroadenine in DNA

8-oxo-7,8-dihydroadenine (8-oxoAde) is a major product of adenine modification by reactive oxygen species. So far, only one mammalian DNA glycosylase, 8-oxoguanine-DNA-glycosylase 1 (OGG1), has been shown to excise 8-oxoAde, exclusively from pairs with Cyt. We have found that endonuclease VIII-like protein 1 (NEIL1), a mammalian homolog of bacterial endonuclease VIII, can efficiently remove 8-oxoAde from 8-oxoAde:Cyt pairs but not from other contexts. In an in vitro reconstituted system, reactions containing OGG1 produced a fully repaired product, whereas NEIL1 caused an abortive initiation of repair, stopping after 8-oxoAde removal and DNA strand cleavage. This block was partially relieved by polynucleotide kinase/3′-phosphatase. Thus, two alternative routes of 8-oxoAde repair may exist in mammals.     

Genetic variation in DNA repair gene XRCC7 (G6721T) and susceptibility to breast cancer

The human XRCC7 is a DNA double-strand break (DSBs) repair gene, involved in non-homologous end joining (NHEJ). It is speculated that DNA DSBs repair have an important role during development of breast cancer. The human XRCC7 is a NHEJ DSBs repair gene. Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. This polymorphism may regulate splicing and cause mRNA instability. In the present study, we specifically investigated whether common G6721T genetic variant of XRCC7 was associated with an altered risk of breast cancer. The present study included 362 females with breast cancer. Age frequency-matched controls (362 persons) were randomly selected from the healthy female blood donors, according to the age distribution of the cases. Using RFLP-PCR based method, the polymorphism of XRCC7 was determined. The TG (OR=1.20, 95% CI: 0.83-1.74, P=0.320) and TT (OR=1.01, 95% CI: 0.67-1.53, P=0.933) genotypes had no significant effect on risk of breast cancer, in comparison with the GG genotype. Our present findings indicate that the TT and TG genotypes were not associated with an altered breast cancer risk.     

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

A new mutation in the gene ROR2 causes brachydactyly type B1

Brachydactyly type B, an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails, can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is caused by mutations in the receptor tyrosine kinase gene ROR2, which maps to chromosome 9q22, whereas BDB2 is caused by point mutations in the bone morphogenetic protein antagonist NOGGIN. Here, we report a three-generation Chinese family with dominant inheritance of the BDB1 limb phenotype. Sequence analysis identified a novel heterozygous base deletion (c.1396–1398delAA) in the gene ROR2 in all affected family members. This new deletion is expected to produce a truncated Ror2 protein with a new polypeptide of 57 amino acids at the C-terminal.     

Trisomy of the Dscr1 gene suppresses early progression of pancreatic intraepithelial neoplasia driven by oncogenic Kras

Individuals with Down syndrome exhibit remarkably reduced incidence of most solid tumors including pancreatic cancer. Multiple mechanisms arising from the genetic complexity underlying Down syndrome has been suggested to contribute to such a broad cancer protection. In this study, utilizing a genetically engineered mouse model of pancreatic cancer, we demonstrate that trisomy of the Down syndrome critical region-1 (Dscr1), an endogenous calcineurin inhibitor localized on chromosome 21, suppresses the progression of pancreatic intraepithelial neoplasia-1A (PanIN-1A) to PanIN-1B lesions without affecting the initiation of PanIN lesions mediated by oncogenic Kras^G12D. In addition, we show that Dscr1 trisomy attenuates nuclear localization of nuclear factor of activated T-cells (NFAT) accompanied by upregulation of the p15^Ink4b tumor suppressor and reduction of cell proliferation in early PanIN lesions. Our data suggest that attenuation of calcineurin–NFAT signaling in neoplastic pancreatic ductal epithelium by a single extra copy of Dscr1 is sufficient to inhibit the progression of early PanIN lesions driven by oncogenic Kras, and thus may be a potential mechanism underlying reduced incidence of pancreatic cancer in Down syndrome individuals.     

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8^AN015 or CSN5^null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75

Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.     

Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

Identification and expression of microRNA in the brain of hibernating bats, Myotis lucifugus

Recent research has highlighted roles for non-coding RNA i7n the regulation of stress tolerance in bats. In this study, we propose that microRNA could also play an important role in neuronal maintenance during hibernation. To explore this possibility, RT-PCR was employed to investigate the expression of eleven microRNAs from the brain tissue of euthermic control and torpid bats. Results show that eight microRNAs (miR-21, -29b, -103, -107, -124a, -132, -183 and -501) increased (1.2–1.9 fold) in torpid bats, while the protein expression of Dicer, a microRNA processing enzyme, did not significantly change during torpor. Bioinformatic analysis of the differentially expressed microRNA suggests that these microRNAs are mainly involved in two processes: (1) focal adhesion and (2) axon guidance. To determine the extent of microRNA sequence conservation in the bat, we successfully identified bat microRNA from sequence alignments against known mouse (Mus musculus) microRNA. We successfully identified 206 conserved pre-microRNA sequences, leading to the identification of 344 conserved mature microRNA sequences. Sequence homology of the identified sequences was found to be 94.76 ± 3.95% and 98.87 ± 2.24% for both pre- and mature microRNAs, respectively. Results suggest that brain function related to the differentiation of neurons and adaptive neuroprotection may be under microRNA control during bat hibernation.