Support DNA Synthesis Research Articles

Unveiling the Therapeutic Potential of Folate-Dependent One-Carbon Metabolism in Cancer and Neurodegeneration.

Cellular metabolism is crucial for various physiological processes, with folate-dependent one-carbon (1C) metabolism playing a pivotal role. Folate, a B vitamin, is a key cofactor in this pathway, supporting DNA synthesis, methylation processes, and antioxidant defenses. In dividing cells, folate facilitates nucleotide biosynthesis, ensuring genomic stability and preventing carcinogenesis. Additionally, in neurodevelopment, folate is essential for neural tube closure and central nervous system formation. Thus, dysregulation of folate metabolism can contribute to pathologies such as cancer, severe birth defects, and neurodegenerative diseases. Epidemiological evidence highlights folate's impact on disease risk and its potential as a therapeutic target. In cancer, antifolate drugs that inhibit key enzymes of folate-dependent 1C metabolism and strategies targeting folate receptors are current therapeutic options. However, folate's impact on cancer risk is complex, varying among cancer types and dietary contexts. In neurodegenerative conditions, including Alzheimer's and Parkinson's diseases, folate deficiency exacerbates cognitive decline through elevated homocysteine levels, contributing to neuronal damage. Clinical trials of folic acid supplementation show mixed outcomes, underscoring the complexities of its neuroprotective effects. This review integrates current knowledge on folate metabolism in cancer and neurodegeneration, exploring molecular mechanisms, clinical implications, and therapeutic strategies, which can provide crucial information for advancing treatments.

Associations between Folate and Alcohol Consumption with Colorectal Tumor Ki67 Expression in the Southern Community Cohort Study

Folate is hypothesized to accelerate cell proliferation in colorectal cancer (CRC) by supporting DNA synthesis, while alcohol is also linked to gastrointestinal epithelial proliferation, despite biological antagonism of folate. We report associations between folate and alcohol consumption with the proliferation marker Ki67 in CRC tumors from the Southern Community Cohort Study. Tumor samples were obtained from formalin-fixed paraffin-embedded tissue blocks. The percentage of cells expressing Ki67 was measured immunohistochemically. Exposures were assessed via questionnaire pre-diagnosis. Associations were assessed via linear regression. In 248 cases (40–78 years), neither dietary folate, folic acid supplements, nor total folate intake were associated with Ki67. Folic acid supplement use was associated with Ki67 in distal/rectal tumors (β [95% confidence interval]: 7.5 [1.2-13.8], p = .02) but not proximal tumors (-1.4 [-7.1-4.3], p=.62). A positive trend for total folate was observed for distal/rectal tumors (1.6 [0.0-3.3] per 200 μcg, p-trend=.05). Heavy drinking (women: ≥1 drink/day, men: ≥2 drinks/day) was associated with higher Ki67 (6.4 [1.0-11.9], vs. nondrinkers, p=.02), especially for distal/rectal tumors (10.4 [1.6-19.1], p=.02). Negative interaction between alcohol, total folate was observed for distal/rectal tumors (p-interaction=.06). Modest associations between folate, alcohol consumption and distal/rectal tumor Ki67 expression suggest accelerated proliferation, consistent with folate’s role in DNA synthesis.

The N‐terminal Domain of Twinkle, the Replicative Helicase in Human Mitochondria, Binds DNA and is Essential in Supporting Processive DNA Synthesis by the Mitochondrial DNA Polymerase

Twinkle is the replicative helicase in human mitochondria, and is relatively poorly studied compared to its nuclear counterpart. Evolutionarily related helicases in the SF4 family, including T7 gp4 and Twinkle orthologs have an N‐terminal domain that serve as the primase for replication while Human Twinkle has been demonstrated to have lost this function, with several key metal binding residues being lost evolutionarily. Without the primase activity, the utility of the NTD is in question as Human Twinkle has been demonstrated to retain its helicase functionality without the NTD, with somewhat reduced efficiency. We generated and purified a novel Twinkle construct containing only the C‐terminal domain and linker region (CTD, amino acids 360‐684) alongside a full length (aa 43‐684) and a previously studied N‐terminal domain construct (NTD, aa 43‐372). The NTD does not oligomerize in gel filtration analysis or have any of the enzymatic activities of Twinkle. Interestingly, NTD displays a notable nonspecific binding affinity for nucleic acids longer than 16 nt. The CTD retains much of the activities of full‐length Twinkle including comparable DNA binding and, annealing, and somewhat reduced ATPase and helicase activities. CTD supports strand displacement synthesis by Polymerase γ on short replication forks but does not promote formation of multi‐kilobase length products in rolling circle DNA synthesis. Strikingly, CTD is able to support long stretch DNA replication when it is part of the reconstituted mitochondrial replisome with Polymerase γ and mtSSB, albeit with a lower efficiency than Human Twinkle. Thus, the C‐terminal domain retains the majority of the lower order component abilities of full length Twinkle but struggles to implement them for processive DNA synthesis. The results suggest that the role of the N‐terminal domain of Twinkle is in optimizing the motor activity of the C‐terminal domain for synthesizing biologically relevant lengths of DNA. The implications are foundational for understanding the mechanism of action for a subsection of sometimes debilitating mitochondrial genetic diseases which result from several single point mutations in the N‐terminal region which have previously been cryptic.

The Roles of Mitochondrial Folate Metabolism in Supporting Mitochondrial DNA Synthesis, Oxidative Phosphorylation, and Cellular Function.

ABSTRACTFolate-mediated one-carbon metabolism (FOCM) is compartmentalized within human cells to the cytosol, nucleus, and mitochondria. The recent identifications of mitochondria-specific, folate-dependent thymidylate [deoxythymidine monophosphate (dTMP)] synthesis together with discoveries indicating the critical role of mitochondrial FOCM in cancer progression have renewed interest in understanding this metabolic pathway. The goal of this narrative review is to summarize recent advances in the field of one-carbon metabolism, with an emphasis on the biological importance of mitochondrial FOCM in maintaining mitochondrial DNA integrity and mitochondrial function, as well as the reprogramming of mitochondrial FOCM in cancer. Elucidation of the roles and regulation of mitochondrial FOCM will contribute to a better understanding of the mechanisms underlying folate-associated pathologies.

The DNA replication fork suppresses CMG unloading from chromatin before termination.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

Autophagy role(s) in response to oncogenes and DNA replication stress

Autophagy is an evolutionarily conserved process that captures aberrant intracellular proteins and/or damaged organelles for delivery to lysosomes, with implications for cellular and organismal homeostasis, aging and diverse pathologies, including cancer. During cancer development, autophagy may play both tumour-supporting and tumour-suppressing roles. Any relationships of autophagy to the established oncogene-induced replication stress (RS) and the ensuing DNA damage response (DDR)-mediated anti-cancer barrier in early tumorigenesis remain to be elucidated. Here, assessing potential links between autophagy, RS and DDR, we found that autophagy is enhanced in both early and advanced stages of human urinary bladder and prostate tumorigenesis. Furthermore, a high-content, single-cell-level microscopy analysis of human cellular models exposed to diverse genotoxic insults showed that autophagy is enhanced in cells that experienced robust DNA damage, independently of the cell-cycle position. Oncogene- and drug-induced RS triggered first DDR and later autophagy. Unexpectedly, genetic inactivation of autophagy resulted in RS, despite cellular retention of functional mitochondria and normal ROS levels. Moreover, recovery from experimentally induced RS required autophagy to support DNA synthesis. Consistently, RS due to the absence of autophagy could be partly alleviated by exogenous supply of deoxynucleosides. Our results highlight the importance of autophagy for DNA synthesis, suggesting that autophagy may support cancer progression, at least in part, by facilitating tumour cell survival and fitness under replication stress, a feature shared by most malignancies. These findings have implications for better understanding of the role of autophagy in tumorigenesis, as well as for attempts to manipulate autophagy as an anti-tumour therapeutic strategy.

Alternative divalent cations (Zn²⁺, Co²⁺, and Mn²⁺) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.

BackgroundFidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn2+, Co2+, and Zn2+ can also support catalysis. Although Zn2+ supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn2+ is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn2+.MethodsWe used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn2+, Co2+, and Zn2+.ResultsThe fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn2+ when compared to Mg2+ at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3′ nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn2+ than Mg2+. In agreement with previous literature, we observed that Mn2+ and Co2+ dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn2+ and Co2+ remained similar to Mg2+ at lower concentrations that are optimal for catalysis.ConclusionThis study shows that Zn2+, at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

Metabolism: Growth in the fat lane.

Analysis of endothelial cells, which are involved in blood-vessel formation, unexpectedly reveals that proliferation in this cell type depends on fatty-acid oxidation to support DNA synthesis. See Article p.192 Peter Carmeliet and colleagues identify a crucial role for the oxidation of fatty acids in endothelial cells during angiogenesis. They show that fatty acids provide the carbons for the de novo synthesis of nucleotides, and hence fatty acid oxidation stimulates vessel sprouting by increasing endothelial cell proliferation. Pharmacological blockade of fatty acid oxidation can reduce pathological angiogenesis in a mouse model of retinopathy of prematurity.

Pyruvate Kinase Isoform Expression Alters Nucleotide Synthesis to Impact Cell Proliferation

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy

AIDS Research and Human RetrovirusesVol. 30, No. 3 Images in HIV ResearchFree AccessUnraveling the Gymnastics of Reverse Transcription Through Single Molecule SpectroscopyElio A. Abbondanzieri and Stuart F.J. Le GriceElio A. AbbondanzieriDepartment of Bionanoscience, Delft University of Technology, Delft, The Netherlands.Search for more papers by this author and Stuart F.J. Le GriceRT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland.Search for more papers by this authorPublished Online:3 Mar 2014https://doi.org/10.1089/aid.2014.0023AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Synthesizing integration-competent, double-stranded DNA from the single-stranded human immunodeficiency virus (HIV) RNA genome might more aptly be described as molecular gymnastics (Fig. 1). Key elements of this routine include intramolecular and intermolecular transfer of the replication complex between genome termini, strand displacement synthesis, selecting plus-strand RNA primers from within the RNA/DNA replication intermediate, their excision from nascent DNA, and phased A-tract-induced cessation of DNA synthesis near the center of the genome. While site-directed mutagenesis identifies residues of HIV-1 reverse transcriptase (RT) that mediate these functions, and X-ray crystallography gives elegant, but static snapshots of nucleoprotein complexes, they cannot assess conformational dynamics of this remarkably versatile enzyme. To better understand how HIV-1 RT interacts with nucleic acid required designing a single molecule strategy based on Foerster resonance energy transfer (FRET), placing fluorophores into the enzyme and its nucleic acid substrates.FIG. 1. Multistep acrobatics and reverse transcription.Studies with duplex DNA predictably recapitulated an enzyme with its DNA polymerase catalytic center straddled over the primer terminus. However, an unexpected observation of Abbondanzieri et al.1 was that this orientation reversed when the DNA primer was replaced with RNA. Later studies examining polypurine tract recognition not only confirmed that HIV-1 RT could assume different orientations, but also was sufficiently agile to “flip” between these during a single binding event. Such reversal in RT orientation could help explain how its N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) domains can recognize the PPT primer terminus at different steps of plus-strand synthesis.Another concept we accept when studying HIV-1 RT is that it is a distributive polymerase, i.e., it frequently dissociates from its nucleic acid substrate. How then, upon rebinding, does the enzyme locate the primer terminus to reengage in DNA synthesis? Subsequent studies of Liu et al. illustrated the capacity of RT to slide on the nucleic acid duplex, a property that could be enhanced in the presence of nonnucleoside RT inhibitors or restricted by forming a ternary complex. However, the story does not end here. Since there appears to be no orientation (or directional) bias during sliding, the polymerization target site could be accessed by the retroviral polymerase in two different orientations. Single molecule spectroscopy not only confirmed this, but elegantly demonstrated that an enzyme accessing the primer terminus in an “RNase H mode” could spontaneously flip into the polymerization mode, thereby linking the properties of flipping and sliding.2A state-of-the-art technology, high-profile publications, but what is the significance? For many years, we have presumed that nonnucleoside inhibition of HIV-1 RT occurred through a “grinding of the gears” and an inability to support DNA synthesis. However, examining the interaction of HIV-1 RT with its PPT by single molecule spectroscopy revealed that the nonnucleoside reverse transcriptase inhibitor (NNRTI) nevirapine promotes a reversal of enzyme orientation, positioning the RNase H domain over the primer terminus, thereby inhibiting synthesis of plus-strand DNA. These and related studies on tRNA-primed initiation of minus-strand DNA synthesis3 show that, in a different sense, HIV fitness requires nimble and flexible maneuvering of its DNA polymerase!References1 Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang J, Le Grice SFJ, and Zhuang X: Dynamic binding orientations determine the enzymatic activity of HIV reverse transcriptase. Nature 2008;453:184–189. Crossref, Medline, Google Scholar2 Liu S, Abbondanzieri EA, Rausch JW, Le Grice SFJ, and Zhuang X: Slide into action dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 2008;322:1092–1097. Crossref, Medline, Google Scholar3 Liu S, Harada BT, Miller JT, Le Grice SFJ, and Zhuang X: Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 2010;17:1453–1460. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 30Issue 3Mar 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Elio A. Abbondanzieri and Stuart F.J. Le Grice.Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy.AIDS Research and Human Retroviruses.Mar 2014.209-210.http://doi.org/10.1089/aid.2014.0023Published in Volume: 30 Issue 3: March 3, 2014PDF download

G1/S and G2/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G(1) and mitosis. In fact, delaying either the G(1)/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.

Cooperation between Catalytic and DNA Binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Prognostic significance of serum thymidine kinase activity (TK1) level in patients with metastatic non-small cell lung cancer (NSCLC).

e18010 Background: TK is an enzyme supporting DNA synthesis under conditions of increased cell proliferation. The prognostic significance of TK1 activity in hematological malignancies and early stage solid cancers are well studied but little is known about the prognostic significance of TK1 activity in metastatic NSCL cancer. Methods: TK1 activity was measured by means of novel high sensitive non radioactive DIVITUM assay. We prospectively measured serum TK1 activity immediately before the first and second cycle of the treatment in 48 consecutive metastatic NSCL cancer patients. 10 healthy volunteers were also included as a control group. We aimed to evaluate the value of prognostic significance of TK1 activity. In addition, the clinical significance of the difference between the two serum TK1 activity levels was also assessed. Results: The mean TK1 level in the study group was significantly higher than the controls (respectively 325.6±63.46 vs. 32.97±7.307; p<0.0001). TK1 levels were positively correlated with weight loss (p=0.039). TK1 activity levels in the squamous subtype were higher than the adenocarcinoma but this difference was not significant. (respectively 444.2 ± 143.4 vs. 225.1 ± 59.77; p=0.108). A cut off value of 156 Du/l (AUC 0.77 %95 CI 0.62-0.92; sensitivity %56,1-specifity %14) was determined by ROC curve analysis. High TK1 values were associated with decreased PFS and OS. In multivariate analysis TK1 levels continued to be an independent prognostic factor for patients with metastatic NSCLC (p value for OS was found to be 0.02). Although the difference was not clinically significant, the mean TK1 levels before second cycle of chemotherapy was higher than baseline TK1 levels before the start of chemotherapy (respectively 620±35.3Du/L vs. 325.6±63.46 Du/l p). Conclusions: TK1 levels serve as a good independent prognostic factor in the metastatic setting for NSCLC. Early evaluation of TK1 levels during chemotherapy can not be used for monitoring treatment response. In addition TK1 can be a valuable molecular molecular therapeutic target for metastatic NSCLC.

Abstract LB-367: New evidence supports the potential use of methotrexate in children with MYCN-amplified neuroblastoma.

Abstract Background Folate is essential for cell growth and differentiation. Cancer cells frequently up-regulate folate receptors to increase folate uptake to meet their elevated need for nucleotides to support DNA synthesis and growth. SLC19A1 encodes for a major folate or anti-folate transporter and has been reported to be regulated by oncogenic transcription factors. In childhood neuroblastoma, amplification of the MYCN oncogene is a strong prognostic marker for predicting unfavourable outcome. In light of previous findings showing that overexpression of SLC19A1 increases folate influx and methotrexate (MTX) sensitivity, we aimed to investigate the relationship between SLC19A1 expression, MYCN amplification and expression, to determine if this relationship modified MTX sensitivity in neuroblastoma. Methods Expression levels of SLC19A1 and MYCN were examined in 42 neuroblastoma patient samples and in two independent in vitro MYCN functional systems. The impact of MYCN amplification/expression and SLC19A1 expression on MTX sensitivity were evaluated on 13 neuroblastoma cell lines. SLC19A1 G80A polymorphism was also examined to determine its impact on SLC19A1 gene expression and MTX sensitivity. Results Expression levels of SLC19A1 and MYCN were positively correlated in neuroblastoma tumour samples (p=0.0001). Induction of MYCN in vitro increased the mRNA expression levels of SLC19A1 by 12-fold (p=0.0003) while silencing of MYCN reduced the expression levels of SLC19A1 by 7-fold (p=0.0017). Neuroblastoma cell lines with MYCN overexpression or amplification were at least 2000-fold more sensitive to MTX than those without (expression: p=0.0002; amplification: p&amp;lt;0.0001). However, SLC19A1 G80A polymorphism did not appear to impact on SLC19A1 gene expression or MTX sensitivity. Conclusions Our data indicate a potential functional link between MYCN and the regulation of anti-folate uptake. Our findings suggest that MYCN amplification confers MTX sensitivity through the regulation of the folate receptor; SLC19A1. Targeting SLC19A1 mediated anti-folate uptake in patients with MYCN amplification may provide a new therapeutic approach in children with neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-367. doi:10.1158/1538-7445.AM2011-LB-367

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

The effect of mutation of hepatitis B virus core protein dimer interface domain related to nucleocapsid formation on HBV replication

Objective To investigate the effect of hepatitis B virus core protein (HBc) dimer interfaces amino acids mutation on nucleocapsid assembly and HBV DNA replication. Methods Based on HBc three dimension structure, four HBc dimer interfaces domain mutation plasmids, pHBc14-18M,pHBc120-135M,pHBc23-39M and pHBc122-139M were constructed in pcDNA3.1 vector by PCR site-directed mutagenesis, there was a flag-tag at the C-terminal of all mutants for easy detection. Wild type core protein plasmid 1-183flag was also constructed as a positive control. The 4 mutants were cotransfected HepG2 cells with pHBV1.2 core negative plasmid (pHBV1.2-core-) ,which contained 1.2 copies of HBV whole genome but the core protein would not express due to a stop codon. The capsid formation, HBV pregenome(pgRNA) and HBV DNA replication mediate were analyzed by native agarose gel electrophoresis and Western blot, Northern blot and Southern blot , respectively. The 4 mutants were also cotransfected HepG2 cells with HBV wild type plasmid pHBV1.2 and examined by Southern blot. Virions in the medium were determined by native agarose gel electrophoresis and Western blot. Results Cotransfecting HepG2 cells with pHBV1.2-core- plasmid, pHBc14-18M,pHBc120-135M and pHBc122-139M mutant groups formed nucleocapsid-like structure but pHBc23-39M could not, Northern and Southern blot displayed no signal in all mutants except 1-183flag conrol group. In pHBV1.2 cotransfection experiment, HBV DNA replication was blocked in pHBc14-18M, pHBc120-135M and pHBc122-139M mutant groups, sharply decreased in pHBc120-135M and pHBc122-139M groups, correspondingly virons production in medium were also inhibited. pHBc23-39M mutant exerted no influence on HBV replication. Conclusion pHBc23-39M mutant can neither form nucleocapsid-like structure nor interact with wild type HBc dimmer to interfere HBV replication.On the contrast, pHBc14-18M, pHBc120-135M and pHBc122-139M mutants can form nucleocapsid-like structure by themselves, but this structure does not support HBV DNA synthesis. Besides, they can effectively inhibit wild type HBV DNA replication by contacting with wild HBc dimmers resulting in nucleocapsid dysfunction. Key words: hepatitis B virus; HBc; Core particle assembly; Mutants

An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro

Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells.

Characterization of the contribution of spliced RNAs of hepatitis B virus to DNA synthesis in transfected cultures of Huh7 and HepG2 cells

Hepatitis B virus synthesizes multiple spliced RNAs that can be reverse transcribed into viral DNA. We thoroughly characterized the contribution of spliced RNAs to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. We found that up to 50% of DNA within intracellular capsids is derived from five spliced RNAs. Expressing HBV P protein and pgRNA from separate plasmids and the use of the CMV-IE promoter contributes to these high levels of encapsidated DNA derived from spliced RNA. A spliced RNA called Sp1 was the predominant species expressed in both cell lines. All spliced RNAs support the synthesis minus-strand DNA and duplex linear DNA. Only one of the spliced RNAs, Sp14, supported the synthesis of relaxed circular DNA because splicing removed an important cis-acting sequence (hM) in the other four RNAs. Additionally, we created a variant that was deficient in the synthesis of spliced RNA and supported DNA synthesis at wild-type levels. Our results reinforce and extend the idea that a significant fraction of HBV DNA synthesized under common experimental conditions is derived from spliced RNA. It is important that their presence be considered when analyzing HBV DNA replication in transfected cell cultures.

Overview on Pathophysiology and Newer Approaches to Treatment of Peripheral Neuropathies

Peripheral neuropathies are extremely heterogeneous nosological entities. One of the most common symptoms is pain, the underlying mechanisms of which are numerous and complex. Inflammation, reparative processes, and anatomical and gene expression alterations lead to chronic pain, the persistence of which is sustained by peripheral and central sensitisation mechanisms. Treatment of peripheral neuropathies is targeted to its symptomatic and aetiological features. For pain relief, several types of drugs may be used, notably antidepressants (e.g. tricyclic antidepressants, selective serotonin reuptake inhibitors, and both serotonin and noradrenaline [norepinephrine] reuptake inhibitors), antiepileptic drugs (e.g. carbamazepine, phenytoin, lamotrigine, valproic acid, gabapentin, topiramate and pregabalin), NSAIDs and opioid analgesics. Aetiological therapy is aimed at modifying the pathophysiological mechanisms underlying the neuropathy, some of which are common in different neuropathic conditions. Certain drugs are known to exert more than one action on different pathophysiological mechanisms. This is the case with acetyl-L-carnitine (ALC), which can be considered both a symptomatic therapy that can be used in any kind of painful neuropathy, and an aetiological therapy, at least in diabetic neuropathy and neuropathies induced by nucleoside reverse transcriptase inhibitors and cancer chemotherapeutic agents. ALC acts via several mechanisms, inducing regeneration of injured nerve fibres, reducing oxidative stress, supporting DNA synthesis in mitochondria, and enhancing nerve growth factor concentrations in neurons.

Biochemical and Genetic Analysis of the Vaccinia Virus D5 Protein: Multimerization-Dependent ATPase Activity Is Required To Support Viral DNA Replication

The vaccinia virus-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive (ts) D5 mutants (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7 degrees C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7 degrees C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive Cts24 infections, only a wtD5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA+ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA+ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.