Biochemical Activity of 17 Cancer-Associated Variants of DNA Polymerase Kappa Predicted by Electrostatic Properties.

DNA damage and repair have been widely studied in relation to cancer and therapeutics. Y-family DNA polymerases can bypass DNA lesions, which may result from external or internal DNA damaging agents, including some chemotherapy agents. Overexpression of the Y-family polymerase human pol kappa can result in tumorigenesis and drug resistance in cancer. This report describes the use of computational tools to predict the effects of single nucleotide polymorphism variants on pol kappa activity. Partial Order Optimum Likelihood (POOL), a machine learning method that uses input features from Theoretical Microscopic Titration Curve Shapes (THEMATICS), was used to identify amino acid residues most likely involved in catalytic activity. The µ4 value, a metric obtained from POOL and THEMATICS that serves as a measure of the degree of coupling between one ionizable amino acid and its neighbors, was then used to identify which protein mutations are likely to impact the biochemical activity. Bioinformatic tools SIFT, PolyPhen-2, and FATHMM predicted most of these variants to be deleterious to function. Along with computational and bioinformatic predictions, we characterized the catalytic activity and stability of 17 cancer-associated DNA pol kappa variants. We identified pol kappa variants R48I, H105Y, G147D, G154E, V177L, R298C, E362V, and R470C as having lower activity relative to wild-type pol kappa; the pol kappa variants T102A, H142Y, R175Q, E210K, Y221C, N330D, N338S, K353T, and L383F were identified as being similar in catalytic efficiency to WT pol kappa. We observed that POOL predictions can be used to predict which variants have decreased activity. Predictions from bioinformatic tools like SIFT, PolyPhen-2, and FATHMM are based on sequence comparisons and therefore are complementary to POOL but are less capable of predicting biochemical activity. These bioinformatic and computational tools can be used to identify SNP variants with deleterious effects and altered biochemical activity from a large data set.

Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2'-deoxyguanosine Translesion Synthesis.

Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2'-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Development of Staphylococcus aureus tolerance to antimicrobial photodynamic inactivation and antimicrobial blue light upon sub-lethal treatment.

Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are considered low-risk treatments for the development of bacterial resistance and/or tolerance due to their multitargeted modes of action. In this study, we assessed the development of Staphylococcus aureus tolerance to these phototreatments. Reference S. aureus USA300 JE2 was subjected to 15 cycles of both sub-lethal aPDI (employing an exogenously administered photosensitizer (PS), i.e., rose Bengal (RB)) and sub-lethal aBL (employing endogenously produced photosensitizing compounds, i.e., porphyrins). We demonstrate substantial aPDI/aBL tolerance development and tolerance stability after 5 cycles of subculturing without aPDI/aBL exposure (the development of aPDI/aBL tolerance was also confirmed with the employment of clinical MRSA and MSSA strain as well as other representatives of Gram-positive microbes, i.e. Enterococcus faecium and Streptococcus agalactiae). In addition, a rifampicin-resistant (RIFR) mutant selection assay showed an increased mutation rate in S. aureus upon sub-lethal phototreatments, indicating that the increased aPDI/aBL tolerance may result from accumulated mutations. Moreover, qRT-PCR analysis following sub-lethal phototreatments demonstrated increased expression of umuC, which encodes stress-responsive error-prone DNA polymerase V, an enzyme that increases the rate of mutation. Employment of recA and umuC transposon S. aureus mutants confirmed SOS-induction dependence of the tolerance development. Interestingly, aPDI/aBL-tolerant S. aureus exhibited increased susceptibility to gentamicin (GEN) and doxycycline (DOX), supporting the hypothesis of genetic alterations induced by sub-lethal phototreatments. The obtained results indicate that S. aureus may develop stable tolerance to studied phototreatments upon sub-lethal aPDI/aBL exposure; thus, the risk of tolerance development should be considered significant when designing aPDI/aBL protocols for infection treatments in vitro and in clinical settings.

DNA repair in plant mitochondria - a complete base excision repair pathway in potato tuber mitochondria.

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

High-dose Valganciclovir Treatment for Resistant Cytomegalovirus Colitis due to UL97 and UL54 Mutations.

We report the first case of a ganciclovir-resistant cytomegalovirus (CMV) involving the gastrointestinal tract that was successfully treated with high-dose valganciclovir. A kidney transplant recipient developed drug-resistant CMV colitis which was initially treated with valganciclovir, but his CMV was found to have major resistance to ganciclovir and cidofovir due to UL97 and UL54 mutations. The patient was switched to intravenous foscarnet 40 mg/kg given every twelve hours. However, foscarnet had to be discontinued after 4 days of treatment due to acute kidney injury. Patient was restarted on valganciclovir at a higher target dose of 1800 mg twice a day based on the creatinine clearance. CMV became undetectable 2 weeks after valganciclovir treatment was completed. High-dose valganciclovir along with immune suppression reduction may be a treatment option for CMV colitis with ganciclovir resistance due to dual UL97 and UL54 gene mutations.

Polµ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Polλ and Polµ. It is now well established that Polµ is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.

Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients.

Background: Antiviral-resistant herpes simplex virus type 1 (HSV-1) has been recognized as an emerging clinical problem among patients undergoing hematopoietic stem cell transplantation (HSCT). Methods: A prospective observational study was conducted at a hematological center over a 2-year period. Oropharyngeal swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation. The HSV-1 isolates were tested for sensitivity to acyclovir (ACV). The prognosis of patients with ACV-resistant (ACVr) HSV-1 and the genetic background of the ACVr HSV-1 isolates were assessed. Results: Herpes simplex virus type 1 was isolated in 39 of 268 (15%) HSCT patients within 100 days after transplantation. Acyclovir-resistant HSV-1 emerged in 11 of these 39 patients (28%). The 100-day death rates of HSCT patients without HSV-1 shedding, those with only ACV-sensitive HSV-1 shedding, and those with ACVr HSV-1 shedding were 31%, 39%, and 64%, respectively. Patients with HSV-1, including ACVr HSV-1, shedding showed a significantly higher mortality rate. Relapsed malignancies were a significant risk factor for the emergence of ACVr HSV-1. Acyclovir resistance was attributable to viral thymidine kinase and DNA polymerase mutations in 6 and 5 patients, respectively. Conclusions: Herpes simplex virus type 1, including ACVr HSV-1, shedding was associated with poorer outcome in HSCT patients, even if HSV disease did not always occur. Patients with relapsed malignancies were at especially high risk for the emergence of ACVr HSV-1.

Fidelity and representativeness of two isothermal multiple displacement amplification systems to preamplify limiting amounts of total RNA.

In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst, respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 to 100 ng of starting RNA. The average yield obtained from 10 ng was 3.5 and 4.7 µg for WTA-Phi and WTA-Bst, respectively, while 100 ng of starting RNA yielded 7.0 and 12.4 µg for WTA-Phi and WTA-Bst, respectively. Representational distortion of the templates, analyzed via conventional PCR, showed robust amplification of 11 different transcripts when either WTA-Phi or WTA-Bst synthesized templates were used, while some transcripts were not detected from unamplified templates. Loci representation, a measure of amplification consistency, was evaluated using TaqMan RT-qPCR amplification of five different transcripts, yielding values ranging from 96.4 to 189.3 %, comparable to those obtained using genomic target-based MDA systems. The two MDA protocols described in this study efficiently lead to representative WTA, using as little as 10 ng of starting RNA.

[Diagnostic and therapeutic strategy for acyclovir-resistant herpes encephalitis].

Acyclovir (ACV), which inhibits the replication of herpes simplex virus, is the standard drug for the treatment of herpes simplex encephalitis. Thanks to the introduction of ACV, the morbidity and mortality of HSE patients have significantly improved. However, the disease is still the severe infection, because it makes some patients with HSE suffer from severe consequences. The sensitivity test of the etiological HSV to ACV is very difficult due to the inability of isolation of the virus from cerebrospinal fluid (CSF). The cases of the ACV treatment-resistant HSE patients have been reported. However, these cases were not virologically confirmed. The first case of encephalitis in newborn baby with HSE caused by an ACV-resistant HSV-1, which was virologically confirmed, was reported by our group. According to the sensitivity profile of the causative viruses to antiviral drugs, the drugs of choice for HSE should be properly considered. Strategy for diagnoses of HSE including antiviral sensitivity assessment and selection of drugs in HSE is reviewed.

Phenotypic and genotypic characterization of acyclovir-resistant corneal HSV-1 isolates from immunocompetent patients with recurrent herpetic keratitis.

Herpes simplex virus type 1 (HSV-1) is a leading cause of corneal blindness. Acyclovir (ACV) constitutes the standard treatment of HSV infections including herpetic keratitis (HK). HSV resistance to ACV is mainly described in immunocompromised patients. We describe two cases of ACV-resistant corneal HSV-1 in immunocompetent individuals with recurrent HK.

Cycloartenyl trans-ferulate, a component of the bran byproduct of sake-brewing rice, inhibits mammalian DNA polymerase and suppresses inflammation.

During the screening of selective DNA polymerase (pol) inhibitors, we isolated cycloartenyl trans-ferulate (CAF), which is a major component of γ-oryzanol, which is a byproduct formed during the production of Japanese rice wine "sake". CAF selectively inhibited the activity of mammalian A, B, and X pol families, but Y family pols were not affected. CAF did not influence the activities of plant or prokaryotic pols, nor the activity of other DNA metabolic enzymes tested. Individual chemical components of CAF, including cycloartenol (CA) and ferulic acid (FA), did not inhibit pol enzyme activities. CAF suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in the mouse ear, but CA and FA did not. The ability to inhibit mammalian pol enzymes in vitro was positively correlated with their propensity to suppress inflammation in vivo. These results suggest that this byproduct formed during the sake-brewing process is useful as an anti-inflammatory agent.

Allitridin inhibits human cytomegalovirus replication in vitro.

Human cytomegalovirus (HCMV) has been associated with a wide spectrum of diseases. There is currently no effective treatment for eliminating the virus. Garlic bulb extract has been reported to possess anti-viral efficacy. This study aimed to investigate the expression of the immediate­early (IE; ul122 and ul123), early (E; ul54) and late (L; ul83) genes of HCMV as well as the inhibitory effect of allitridin on the transcription levels of these genes. The results indicated that a HCMV gene expression cascade occurred, and that the deletion of IE72 had no influence on the transcription of the ul122 gene, while it led to significant reductions of ul54 and ul83 mRNA expression levels. Additionally, allitridin effectively suppressed the transcription of the HCMV IE, E and L genes; the inhibition rates of the transcription of the ul122 and ul123 genes were higher compared with those of ul54 and ul83 mRNA expression, while the expression of the IE genes was not significantly reduced by ganciclovir (GCV). Our results indicate that the HCMV IE72 deletion mutant strain affects the transcription of the virus downstream gene, allitridin inhibits HCMV infection in vitro, and that the IE genes may be the key target of allitridin in its action against HCMV.

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production.

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological-chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.

[Bioluminescent reporters to identify gene allelic variants].

The method of single nucleotide polymorphism identification based on primer extension reaction (PEXT) with the following bioluminescent solid-phase microassay was developed. The recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent luciferase Renilla muelleri were used as reporters. Factor V Leiden polymorphism 1691 G-->A (R506Q) of human F5 gene genotyping was used for investigation. Genomic DNA was amplified by PCR using primers, flanking polymorphic site of 140 base pairs. PCR products were used as a template for two PEXT reaction using two primers with 3'-end nucleotides, complementary either normal or mutant alleles. At complementarity of template and allelic-typical primer its extension with DNA-polymerase takes place. The products carried biotin due to availability ofbiotinylated dUTP in the reactions mixture. The assay was carried out using obelin-streptavidin chemical conjugates. Optimal PEXT-reaction conditions providing high reliability of SNP genotyping were found. A new approach to determine both alleles in one well was developed applying two bioluminescent reporters. Availability of the proposed approach was shown in the study of clinical DNA samples.

In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA polymerase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV(r)) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Evolution of linear mitochondrial genomes in medusozoan cnidarians.

In nearly all animals, mitochondrial DNA (mtDNA) consists of a single circular molecule that encodes several subunits of the protein complexes involved in oxidative phosphorylation as well as part of the machinery for their expression. By contrast, mtDNA in species belonging to Medusozoa (one of the two major lineages in the phylum Cnidaria) comprises one to several linear molecules. Many questions remain on the ubiquity of linear mtDNA in medusozoans and the mechanisms responsible for its evolution, replication, and transcription. To address some of these questions, we determined the sequences of nearly complete linear mtDNA from 24 species representing all four medusozoan classes: Cubozoa, Hydrozoa, Scyphozoa, and Staurozoa. All newly determined medusozoan mitochondrial genomes harbor the 17 genes typical for cnidarians and map as linear molecules with a high degree of gene order conservation relative to the anthozoans. In addition, two open reading frames (ORFs), polB and ORF314, are identified in cubozoan, schyphozoan, staurozoan, and trachyline hydrozoan mtDNA. polB belongs to the B-type DNA polymerase gene family, while the product of ORF314 may act as a terminal protein that binds telomeres. We posit that these two ORFs are remnants of a linear plasmid that invaded the mitochondrial genomes of the last common ancestor of Medusozoa and are responsible for its linearity. Hydroidolinan hydrozoans have lost the two ORFs and instead have duplicated cox1 at each end of their mitochondrial chromosome(s). Fragmentation of mtDNA occurred independently in Cubozoa and Hydridae (Hydrozoa, Hydroidolina). Our broad sampling allows us to reconstruct the evolutionary history of linear mtDNA in medusozoans.

Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,ß-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.