Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates

Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme's active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei.     

DNA repair pathway genes and lung cancer susceptibility: A meta-analysis

Objective

DNA repair pathway genes have been implicated to play an important role in the development of lung cancer. However, contradictory results are often reported by various studies, making it difficult to interpret them. So in this meta-analysis, we have assessed the association between lung cancer risk and two DNA repair pathway genes. XRCC1 and ERCC2, by analyzing 67 published case–control studies.

Research design and methods

We searched PubMed, Embase and Web of Science using terms “XRCC1” or “XPD” or “ERCC2” and “lung cancer” on August 1, 2012. Three criteria were applied to select included studies for resulting studies. Information was carefully extracted by two investigators independently. We used pooled odds ratio (OR) to assess the effect of a polymorphism, and a dominant model was applied where genotypes that contain the non-reference allele were combined together. All the calculations were performed using STATA version 11.0.

Main outcome measures and results

Three common nonsynonymous polymorphisms in XRCC1, codon 194, codon 280 and codon 399, and two common nonsynonymous polymorphisms in ERCC2, codon 312 and codon 751, were analyzed. The result showed in total population, Lys751Gln in ERCC2 is associated with an increase of lung cancer risk, with a summary OR as 1.15. No association was found for any other polymorphisms. When studies were stratified by ethnicity, the risk effect of Lys751Gln in ERCC2 was found only in Caucasians, not in Asians.

Conclusions

In conclusion, Lys751Gln in ERCC2 is associated with lung cancer, and the risk effect probably exists in Caucasians. By contrast, polymorphisms in XRCC1 are less likely to be susceptible to lung cancer risks.     

A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis

XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent − 1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′₂C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.     

Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization

The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far.Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.     

Selective purification and characterization of adiponectin multimer species from human plasma

Adiponectin is an adipocyte-derived hormone and known to form several species of multimer, however, the precise components of each multimer have not been fully determined. We purified each multimer adiponectin selectively from human plasma and characterized them by affinity columns using anti-adiponectin, gelatin, or anti-albumin antibody and gel filtration. We found that adiponectin exists as four species of multimers in human plasma. According to their migrating mobility and N-terminal amino acid analysis, we defined them as a trimer, albumin-binding trimer, hexamer, and HMW. Low pH shifted HMW to hexamer, raising the possibility that HMW is a 12 mer or larger multimer. We also showed that HMW had the highest binding activity to the membrane fractions of C2C12 myocytes and activated AMPK most potently. Our results indicate that adiponectin forms diverse multimer species and at least some of the functional properties are dependent on a multimer status.     

DNA base repair – recognition and initiation of catalysis

Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR).     

Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, formed during incomplete burning of coal, oil and gas. Several PAHs have carcinogenic and mutagenic potencies, but these compounds must be activated in order to exert their mutagenic effects. One of the principal pathways proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible for the repair of anthanthrene metabolite-induced DNA damage. However, when the NER pathway was inactivated, a residual repair pathway performed the DNA repair.     

Specificity for Homooligomer versus Heterooligomer Formation in Integrin Transmembrane Helices

Transmembrane (TM) helices engage in homomeric and heteromeric interactions that play essential roles in the folding and assembly of TM proteins. However, features that explain their propensity to interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins provide an ideal model system for addressing these questions because the TM helices of full-length integrins interact heteromerically when integrins are inactive, but isolated TM helices are also able to form homodimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homointeractions versus heterointeractions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of integrin αIIbβ3 TM helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, which are located on the same face of the β3 helix, mediate homodimer formation. We then characterized the β3 heterodimer by measuring the ability of β3 helix mutations to cause ligand binding to αIIbβ3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small residue-X₃-small residue motif S699-X₃-A703, caused constitutive αIIbβ3 activation, as well as persistent focal adhesion kinase phosphorylation dependent on αIIbβ3 activation. Because αIIb and β3 use the same face of their respective TM helices for homomeric and heteromeric interactions, the interacting surface on each has an intrinsic “stickiness” predisposing towards helix-helix interactions in membranes. The residues responsible for heterodimer formation comprise a network of interdigitated side chains with considerable geometric complementarity; mutations along this interface invariably destabilize heterodimer formation. By contrast, residues responsible for homomeric interactions are dispersed over a wider surface. While most mutations of these residues are destabilizing, some stabilized homooligomer formation. We conclude that the αIIbβ3 TM heterodimer shows the hallmark of finely tuned heterodimeric interaction, while homomeric interaction is less specific.     

Role of interaction of XPF with RPA in nucleotide excision repair

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5′ incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.     

Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family

AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-γ-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-γ-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.     

Effect of periplasmic expression of recombinant mouse interleukin-4 on hydrogen peroxide concentration and catalase activity in Escherichia coli

Oxidative stress occurs as a result of imbalance between generation and detoxification of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Relation between cytoplasmic recombinant protein expression with H₂O₂ concentration and catalase activity variation was already reported. The periplasmic space of E. coli has different oxidative environment in relative to cytoplasm and there are some benefits in periplasmic expression of recombinant proteins. In this study, hydrogen peroxide concentration and catalase activity following periplasmic expression of mouse IL-4 were measured in E. coli. After construction of pET2mIL4 plasmid, the expression of recombinant mouse interleukin-4 (mIL-4) was confirmed. Then, the H₂O₂ concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H₂O₂ concentration of the cells. However, the H₂O₂ concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H₂O₂ elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H₂O₂ concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase.     

A genetic variant in the promoter of APE1 gene (− 656 T > G) is associated with breast cancer risk and progression in a Chinese population

Background/aims

APE1 is an important DNA repair protein in the base excision repair pathway. Genetic variations in APE1 have been suggested to influence individuals' susceptibility to human malignancies. The present study was aimed to investigate the associations between two functional polymorphisms in APE1 (− 656 T > G and 1349 T>G) and breast cancer risk.

Methods

We genotyped the two polymorphisms in a case-control study of 500 breast cancer patients and 799 age-matched cancer-free controls using the TaqMan method. Unconditional logistic regression adjusted for potential confounding factors was used to assess the associations.

Results

We found that the variant genotypes of the − 656 T>G were significantly associated with decreased breast cancer risk, compared with the wild genotype [TG/GG vs. TT: adjusted odds ratio (OR) = 0.71, 95% confidence interval (CI) = 0.56–0.91], and the protective effect of this polymorphism was more predominant among the subgroups of younger subjects (< 52 years) (OR = 0.65, 95% CI = 0.46–0.92). Besides, we found that the variant genotypes were associated with less frequent lymph node metastasis (P = 0.020, OR = 0.64, 95% CI = 0.44–0.94). We did not observe any significant association between the 1349 T>G polymorphism and breast cancer risk.

Conclusion

Our results suggest that the APE1 − 656 T>G but not the 1349 T>G polymorphism may influence the susceptibility and progression of breast cancer in the Chinese population. Large population-based prospective studies are required to validate these findings.     

XRCC1 polymorphisms and differentiated thyroid carcinoma risk: A meta-analysis

The objective of this study is to quantitatively derive a more precise estimation of the association between X-ray repair cross-complementing group 1 (XRCC1) gene polymorphisms and differentiated thyroid carcinoma risk. A comprehensive literature search of three databases was conducted. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated with fixed-effect models and random-effect models when appropriate. Overall, no association of the XRCC1 Arg399Gln, Arg280His, and Arg194Trp polymorphisms with differentiated thyroid carcinoma risk was found. In subgroup analyses, a decreased differentiated thyroid carcinoma risk was observed among Caucasians (Gln vs. Arg, OR = 0.86, 95% CI = 0.77–0.96, P = 0.343 for heterogeneity; Gln/Arg vs. Arg/Arg, OR = 0.84, 95% CI = 0.71–0.98, P = 0.229 for heterogeneity; Gln/Gln vs. Arg/Arg, OR = 0.77, 95% CI = 0.60–0.99, P = 0.477 for heterogeneity; dominant genetic model, OR = 0.82, 95% CI = 0.71–0.95, P = 0.272 for heterogeneity), not among Asians. No publication bias was observed. Our results suggest that XRCC1 Arg399Gln polymorphism is not associated with differentiated thyroid carcinoma risk, while a decreased risk is observed among Caucasian population.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.