Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity,Conformational, Thermodynamic and Cytotoxic Aspects

Background

Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.

Methodology/Principal Findings

Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.

Conclusions/Significance

Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.     

Investigating the structural impacts of I64T and P311S mutations in APE1-DNA complex: a molecular dynamics approach

Background

Elucidating the molecular dynamic behavior of Protein-DNA complex upon mutation is crucial in current genomics. Molecular dynamics approach reveals the changes on incorporation of variants that dictate the structure and function of Protein-DNA complexes. Deleterious mutations in APE1 protein modify the physicochemical property of amino acids that affect the protein stability and dynamic behavior. Further, these mutations disrupt the binding sites and prohibit the protein to form complexes with its interacting DNA.

Principal Findings

In this study, we developed a rapid and cost-effective method to analyze variants in APE1 gene that are associated with disease susceptibility and evaluated their impacts on APE1-DNA complex dynamic behavior. Initially, two different in silico approaches were used to identify deleterious variants in APE1 gene. Deleterious scores that overlap in these approaches were taken in concern and based on it, two nsSNPs with IDs rs61730854 (I64T) and rs1803120 (P311S) were taken further for structural analysis.

Significance

Different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in Molecular dynamic study reveals that predicted deleterious variants I64T and P311S alters the structure as well as affect the stability of APE1-DNA interacting functions. This study addresses such new methods for validating functional polymorphisms of human APE1 which is critically involved in causing deficit in repair capacity, which in turn leads to genetic instability and carcinogenesis.     

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

Background

The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.

Methodology/Principal Findings

Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.

Conclusion/Significance

From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.     

The DNA repair gene APE1 T1349G polymorphism and risk of gastric cancer in a Chinese population

Background

Apurinic/apyrimidinic endonuclease 1 (APE1) has a central role in the repair of apurinic apyrimidic sites through both its endonuclease and its phosphodiesterase activities. A common APE1 polymorphism, T1349G (rs3136820), was previously shown to be associated with the risk of cancers.

Objective

We hypothesized that the APE1 T1349G polymorphism is also associated with risk of gastric cancer.

Methods

In a hospital-based case-control study of 338 case patients with newly diagnosed gastric cancer and 362 cancer-free controls frequency-matched by age and sex, we genotyped the T1349G polymorphism and assessed its associations with risk of gastric cancer.

Results

Compared with the APE1 TT genotype, individuals with the variant TG/GG genotypes had a significantly increased risk of gastric cancer (odds ratio = 1.69, 95% confidence interval = 1.19–2.40), which was more pronounced among subgroups of aged ≤60 years, male, ever smokers, and ever drinkers. Further analyses revealed that the variant genotypes were associated with an increased risk for diffuse-type, low depth of tumor infiltration (T1 and T2), and lymph node metastasis gastric cancer.

Conclusions

The APE1 T1349G polymorphism may be a marker for the development of gastric cancer in the Chinese population. Larger studies are required to validate these findings in diverse populations.     

Gadd45a is an RNA binding protein and is localized in nuclear speckles

Background

The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids.

Principal Findings

Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function.

Significance

The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.     

Highly Mutagenic Exocyclic DNA Adducts Are Substrates for the Human Nucleotide Incision Repair Pathway

Background

Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N ⁶-ethenoadenine (εA) and 3,N ⁴-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5′ to a damaged base in a DNA glycosylase-independent manner.

Methodology/Principal Findings

Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC₅₀ values in the range of 5–10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates, as expected, DNA fragments containing 5′-terminal ε-base residue.

Conclusions/Significance

The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.     

New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways

Background

Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells.

Methodology/Principal Findings

Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5′ next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts.

Conclusions/Significance

This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.     

Systematic biases in DNA copy number originate from isolation procedures

Background

The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal.

Results

While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing.

Conclusion

These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses.     

Characterization of the single stranded DNA binding protein SsbB encoded in the Gonoccocal Genetic Island

Background

Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins.

Methodology/Principal Findings

In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg²⁺ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity.

Conclusions/Significance

We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.     

Construction of eukaryotic expression vectors encoding CFP-10 and ESAT-6 genes and their potential in lymphocyte proliferation

Background:

Mycobacterium (M.) bovis is the agent of bovine tuberculosis (TB) in a range of animal species, including humans. Recent advances in immunology and the molecular biology of Mycobacterium have allowed identification of a large number of antigens with the potential for the development of a new TB vaccine. The ESAT-6 and CFP-10 proteins of M. bovis are important structural and functional proteins known to be important immunogens.

Methods:

In the current study, the DNAs encoding these genes were utilized in the construction of pcDNA 3.1+/ESAT-6 and pcDNA3.1+/CFP-10 plasmids. After intramuscular injection of BALB/c mice with these plasmids, ESAT-6 and CFP-10 mRNA expression was assessed by RT-PCR. Mice were inoculated and boosted with the plasmids to evaluate their effects on lymphocyte proliferation.

Results:

Our results indicate the plasmids are expressed at the RNA level and can induce lymphocyte proliferation.

Conclusion:

Further study is needed to characterize the effect of these antigens on the immune system and determine whether they are effective vaccine candidates against M. bovis. Key Words: Mycobacterium bovis, DNA vaccine, ESAT-6, CFP-10, PPD, Proliferation assay, BALB/c mice     

Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose

Background

Activation-induced cytidine deaminase (AID) is a B-cell-specific DNA mutator that plays a key role in the formation of the secondary antibody repertoire in germinal center B cells. In the search for binding partners, protein coimmunoprecipitation assays are often performed, generally with agarose beads.

Methodology/Principal Findings

We found that, regardless of whether cell lysates containing exogenous or endogenous AID were examined, one of two mouse AID forms bound to agarose alone.

Conclusions/Significance

These binding characteristics may be due to the known post-translational modifications of AID; they may also need to be considered in coimmunoprecipitation experiments to avoid false-positive results.     

Altered protein expression in gestational diabetes mellitus placentas provides insight into insulin resistance and coagulation/fibrinolysis pathways

Objective

To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).

Methods

We used two-dimensional electrophoresis (2DE) to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC) was performed to examine the cellular location of the proteins expressed in placenta villi.

Results

Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group.

Conclusion

Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.     

The Wall Eclipsing Sign on Pulmonary Artery Computed Tomography Angiography Is Pathognomonic for Pulmonary Artery Sarcoma

Background

The objective of this study was to evaluate the imaging characteristics of pulmonary artery sarcoma (PAS) on pulmonary artery computed tomography angiography (PACTA) that can be used to differentiate between PAS and pulmonary thromboembolic diseases, including chronic thromboembolic pulmonary hypertension (CTEPH) and acute pulmonary embolism (APE).

Methods

The clinical data and imaging characteristics of 12 patients with PAS, 156 patients with CTEPH, and 426 patients with APE who were treated at Beijing Anzhen Hospital from January 2007 to August 2013 were retrospectively analyzed. All patients underwent PACTA before treatment, and the diagnoses of PAS and CTEPH were all confirmed by surgical biopsy.

Results

All 12 PAS patients were initially misdiagnosed and received inappropriate thrombolytic and/or anticoagulant therapy before they were referred for surgical intervention. The mean time from PACTA to surgical intervention was 5.5±3.7 months (range 2–11 months). On PACTA, the PAS lesion always eclipsed the wall of the pulmonary artery before infiltrating outside the pulmonary artery, which was termed the wall eclipsing sign. This sign was observed in all PAS patients but was not observed in any CTEPH or APE patients.

Conclusions

PAS is a rare neoplasm with a poor prognosis, and is easily misdiagnosed as thromboembolic disease. The wall eclipsing sign on PACTA is pathognomonic for PAS, and patients with this sign should be investigated to confirm the diagnosis and should undergo surgical intervention as soon as possible, rather than receiving thrombolytic or anticoagulant therapy.