The impact of function perturbations in Boolean networks

MOTIVATION: A network is said to be robust relative to a certain network characteristic if a small change in network structure does not significantly affect the characteristic. From the perspective of network stability, robustness is desirable; however, from the perspective of intervention to exert influence on network behavior, it is undesirable. For Boolean networks, there are two fundamental types of robustness. One type pertains to perturbing the state of the network and the other to perturbing the rule-based structure. RESULTS: This article explores the impact of function perturbations in Boolean networks from two aspects: (1) analysis: predict the impact on network state transitions and attractors via analytical approaches or identify a perturbation by observing its consequences; (2) synthesis: preserve or modify the network characteristics, especially attractors, by introducing a judicious change to the functions. The results are applied to achieve intervention that structurally alters the network to achieve a more favorable steady-state distribution and to identify the function perturbation that has led to altered observed behavior. The intervention procedure is applied to a WNT5A network to reduce the risk of metastasis in melanoma, and the identification procedure is applied to a Drosophila melanogaster segmentation polarity gene network to identify regulatory function perturbation.     

Plant cytosine-5 DNA methyltransferases: structure, function, and molecular evolution

A detailed analysis of the structure and function, along with evolutionary aspects, of the main plant cytosine-5 DNA methyltransferases (C5-MTases) is presented. The evolutionary relationships between the already known and four candidate plant C5-MTases identified in this work were investigated using the distance, maximum-parsimony, and maximum-likelihood approaches. The topologies of the trees were overall congruent: four monophyletic groups corresponding to the four plant C5-MTase families were clearly distinguished. In addition, sequence analyses of the plant C5-MTase target recognition domain sequences were performed and phylogenetic trees were reconstructed showing that there is good conservation among but not within the plant C5-MTase families. Furthermore, a conserved dipeptide that plays an important role in flipping the target base into the catalytic site of the C5-MTases was identified in all plant C5-MTases under study.     

DNA nucleotide excision-repair synthesis is independent of perturbations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.     

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.     

Structural and dynamic perturbations induced by heme binding in cytochrome b5

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.     

Biological function of DNA methylation

Structural and functional properties of prokaryotic DNA methyltransferases are summarized. The different aspects of the role of DNA methylation which influences DNA-protein interaction in restriction and modification of DNA and in mismatch repair, DNA replication and gene expression are discussed.     

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 ± 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.     

Mechanistic analysis of Xenopus EXO1's function in 5'-strand resection at DNA double-strand breaks

The processing of DNA double-strand breaks (DSBs) into 3' single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5'-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis. Xenopus EXO1 (xEXO1) was found to display strong 5'→3' dsDNA exonuclease activity but no significant ssDNA exonuclease activity. Depletion of xEXO1 caused significant inhibition of 5' strand resection. Co-depletion of xEXO1 and Xenopus DNA2 (xDNA2) showed that these two nucleases act in parallel pathways and by distinct mechanisms. While xDNA2 acts on ssDNA unwound mainly by the Xenopus Werner syndrome protein (xWRN), xEXO1 acts directly on dsDNA. Furthermore, xEXO1 and xWRN are required for both the initiation stage and the extension stage of resection. These results reveal important novel information on the mechanism of 5'-strand resection in eukaryotes.     

Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.     

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.     

Single-molecule studies of DNA replisome function

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.     

DNA 5-Methylcytosine Demethylation Activities of the Mammalian DNA Methyltransferases

Methylation at the 5-position of DNA cytosine on the vertebrate genomes is accomplished by the combined catalytic actions of three DNA methyltransferases (DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintenance enzyme DNMT1. Although several metabolic routes have been suggested for demethylation of the vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive. Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates DNMTs in general, can also act as Ca²⁺ ion- and redox state-dependent active DNA demethylases. This finding suggests new directions for reinvestigation of the structures and functions of these DNMTs, in particular their roles in Ca²⁺ ion-dependent biological processes, including the genome-wide/local DNA demethylation during early embryogenesis, cell differentiation, neuronal activity-regulated gene expression, and carcinogenesis.     

5-Lipoxygenase-mediated Endogenous DNA Damage

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 ± 0.01 to 45.50 ± 4.05 pmol/10⁷ cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2′-deoxyguanosine (HϵdGuo) from 2.41 ± 0.35 to 6.31 ± 0.73 adducts/10⁷ normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HϵdGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HϵdGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HϵdGuo DNA-adduct in CESS cells.PUFAs² can be converted into lipid hydroperoxides enzymatically by the action of LOs (1) and COXs (2) or nonenzymatically by reactive oxygen species (ROS) (3). Arachidonic acid, one of the essential PUFAs present in cell membranes, is released from phospholipids by different phospholipase A₂ isoforms upon diverse physical, chemical, inflammatory, and mitogenic stimuli (4). The free arachidonic acid then serves as a substrate for LOs, COXs, or ROS to produce a variety of lipid hydroperoxides (5, 6). ROS, 12-LO, and 15-LO can also act directly upon arachidonic acid esterified in phospholipids to produce lipid hydroperoxides (7), which are reduced (8), hydrolyzed by phospholipase A₂ (4), and then secreted as the corresponding free HETEs (9). COX-2-mediated (10) and 15-LO-1-mediated (11) metabolism of linoleic acid results in the formation of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, which is rapidly reduced to 13(S)-hydroxy-9,11-(Z,E)-octadecadienoic acid (HODE) and secreted from cells. Arachidonic acid is specifically metabolized by 5-LO into 5(S)-HpETE, which is either reduced to 5(S)-HETE or serves as precursor to the formation of leukotrienes (LTs) (Scheme 1) (12). In contrast, ROS-mediated reactions produce racemic mixtures of all possible regioisomers of HpETEs and 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acids (3) that are subsequently secreted from cells as complex mixtures of racemic HETEs and HODEs. Therefore, the ability to analyze different HETE and HODE enantiomers and regioisomers is important for elucidating specific cellular lipid peroxidation pathways (13).Open in a separate windowSCHEME 1.5-LO-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid.The conversion of arachidonic acid to 5(S)-HpETE by 5-LO is critically dependent upon the presence of FLAP (14). 5-LO and FLAP are expressed primarily in inflammatory cells such as polymorphonuclear leukocytes, monocytes, macrophages, and mast cells (12, 15–17). Therefore, 5-LO-mediated LT formation is thought to play a critical role in inflammation and allergic disorders (18–21). In addition, a number of studies have implicated 5-LO-derived arachidonic acid metabolites as mediators of atherogenesis and heart disease (12, 22, 23). The 5-LO pathway of arachidonic acid metabolism has also been proposed to play a role in prostate and pancreatic cancer (24–26).Lipid hydroperoxides undergo homolytic decomposition into bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal, ONE, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy-2(E)-nonenal (27). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH) (28, 29), DNA, (5, 6), and proteins (30, 31). Our previous in vitro studies characterized the bifunctional electrophiles ONE and 4-hydroperoxy-2(E)-nonenal as major products arising from the homolytic decomposition of 5-LO-derived 5(S)-HpETE (32). Reactions with DNA resulted in the formation of etheno-2′-deoxyguanosine (ϵdGuo) from 4-hydroperoxy-2(E)-nonenal and heptanone-ϵdGuo (HϵdGuo) from ONE (Scheme 1). 5,8-Dioxo-6-octenoic acid, a bifunctional electrophile from the carboxyl terminus of 5(S)-HpETE, gave rise to the novel DNA-adduct carboxypentanone-ϵdGuo (CPϵdGuo)-adduct as shown in Scheme 1.Previous studies have demonstrated that lipid hydroperoxides generated by COX-2 could lead to the formation of endogenous DNA adducts in epithelial cells (6). Cellular 5-LO, like COX-2, synthesizes lipid hydroperoxides on the nuclear membrane. Therefore, it is highly possible that 5-LO could also mediate the formation of lipid hydroperoxide-derived endogenous DNA adducts in cells. CESS is a human lymphoblastic cell line that expresses both 5-LO and FLAP, and they have been used as a model for inflammatory cells to examine the role of 5-LO metabolites in signal transduction (33, 34). In the present study, CESS cells provided an ideal model to elucidate the relationship of 5-LO mediated-lipid peroxidation and DNA-adduct formation in a cellular setting. Stable isotope dilution chiral LC-electron capture (EC) APCI/MRM/MS (13) was used to monitor the concomitant formation of lipid hydroperoxides in the presence of different enzyme stimulator or inhibitors. DNA-adduct formation in the same cells was measured by a stable isotope dilution APCI/MRM/MS method. The powerful tool of chiral lipid analysis enabled us to dissect the complicated lipid peroxidation pathways and to correlate them with endogenous DNA-adduct formation. The results demonstrated that 5-LO-mediated lipid peroxidation could cause HϵdGuo formation in cells. This novel finding provided additional explanation for the previous observation that increased 5-LO activity was associated with cancers and cardiovascular diseases (24–26).     

Analysis of MRE11's function in the 5'-->3' processing of DNA double-strand breaks

The resection of DNA double-strand breaks (DSBs) into 3' single-strand tails is the initiating step of homology-dependent repair pathways. A key player in this process is the MRE11-RAD50-NBS1 complex, but its contribution to and mechanistic role in resection are not well understood. In this study, we took advantage of the Xenopus egg extract system to address these questions. We found that depletion of MRE11 caused a dramatic inhibition of 5'-resection, even for the first nucleotide at the 5'-end. Depletion of Xenopus CtIP also inhibited 5'-strand resection, but this inhibition could be alleviated by excess MRN. Both MRE11 and CtIP could be bypassed by a DNA that carried a 3'-ss-tail. Finally, using purified proteins, we found that MRN could stimulate both the WRN-DNA2-RPA pathway and the EXO1 pathway of resection. These findings provide important insights into the function of MRE11 in 5'-strand resection.     

Structure and function of Y chromosomal DNA

The sequence organization of four different families of Y chromosomal repetitive DNA is characterized at three levels of spatial extension along the Y chromosome of Drosophila hydei. At the lowest level of resolution, DNA blot analysis of Y chromosomal fragments of different lengths and in situ hybridization experiments on metaphase chromosomes demonstrate the clustering of each particular sequence family within one defined region of the chromosome. At a higher level of resolution, family specific repeats can be detected within these clusters by crosshybridization within 10–20 kb long continuous stretches of cloned DNA in EMBL3 phages. At the highest level of resolution, detailed sequence analysis of representative subclones about 1 kb in length reveals a satellite-like head to tail arrangement of family specific degenerated subrepeats as the building scheme common to all four families. Our results provide the first comparative sequence analysis of three novel families of repetitive DNA on the long arm of the F chromosome of D. hydei. Additional data are presented which support the existence of two related subfamilies of repetitive DNA on the short arm of the Y chromosome.