In vitro inhibition of rat liver DNA polymerases α and β by β-blockers

The activity of DNA polymerases α and β, isolated from regenerating rat liver, is inhibited, in a dose-dependent fashion, by the oncogenic β-blocker DL-I-(2-nitro-3-m ethyl-phenoxy) - 3-tert-butylamino-propan-2-ol (ZAM[ 1305) and by non-oncogenic β-blockers DL-l-(2-nitro-5-methyl- phenoxy-3-tert-butylamino-propan-2-ol (ZAMI 1327) and DL-propranolol. The inhibition is due to a reversible interaction of the g-blockers with the two DNA polymerases. The interaction does not involve the template-DNA-binding site or the deoxynucleotide-binding site of the enzyme molecule. The degree of inhibition appears to be related to the hydrophobicity of the aromatic moiety and to the length and/or hydrophilicity of the aliphatic chain of the β-blocker molecule. These results may explain the transient in vivo inhibition of hepatic DNA synthesis observed in female rats treated with ZAMI 1305 or ZAMI 1327.     

Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β

Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m²) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

DNA polymerases β and λ and their roles in cell

Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ.     

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability.     

DNA polymerases β and λ and their roles in DNA replication and repair

DNA-dependent DNA polymerases are the main enzymes that catalyze DNA replication. Higher eukaryotic cells have 19 DNA polymerases with strikingly different properties [1]. Mitochondrial DNA polymerase γ of the A family and most of the nuclear enzymes of the B family are high-fidelity DNA polymerases that are involved not only in genomic DNA replication but also in DNA repair. Among the other 15 proteins, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are also involved in repair, including base excision repair and nonhomologous end joining. Some of them play a specific role in replication of damaged DNA templates. This process is referred to as translesion synthesis (TLS). DNA polymerases β and λ, which belong to the X structural family, are polyfunctional enzymes; their properties and functions are discussed.     

Differential roles of estrogen receptors α and β in control of B-cell maturation and selection

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or β engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or β can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.     

Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro,is regulated by flap endonuclease 1 and DNA ligase 1

Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.     

The heparin-accelerated neutralisation of bovine α and β thrombins by antithrombin III

The heparin-accelerated neutralisation of bovine α and β thrombins has been examined using a peptide substrate H-d-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. α and β thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of α and β thrombin. This work is therefore unabel to confirm reports that α and β thrombins have different heparin sensitives.     

DNA polymerases β and λ as potential participants of TLS during genomic DNA replication on the lagging strand

The main strategy used by pro-and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases β and λ on DNA substrates using mono-or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg²⁺ or Mn²⁺. DNA polymerases β and λ were able to catalyze DNA synthesis across THF only in the presence of Mn²⁺. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase β. As for DNA polymerase λ, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase β. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase λ. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases β and λ on DNA templates containing damage.     

Comparison of α and β keratin in reptiles

Summary The different patterns of keratin formation that have evolved in the class Reptilia are all variations of a common process. In Squamata (snakes and lizards), a sequence of layers composed of or keratin is formed periodically, after which the old epidermal generation is shed. In Chelonia (turtles and tortoises), the epidermis of the shell is composed of only keratin, whereas the skin of the neck and leg is composed exclusively of keratin. Molting in toto does not occur and shedding is a continuous process comparable to that in avian and mammalian epidermis. In Crocodilia (crocodiles, caimans, alligators) there is only a single layer of cornified cells, but the composition of the layer varies in different parts of the scale. The hinge regions have many of the morphological characteristics of and keratin whereas the center resembles keratin. The living cells beneath contain accumulations of keratohyalin.There are four ultrastructural characteristics of a keratinized layer: 1) cellular outlines remain distinct, 2) a thickened plasma membrane forms during keratinization, 3) 80 Å filaments embedded in an amorphous matrix can be seen, and 4) PAS-positive material accumulates in extracellular spaces between the desmosomes.The layer exhibits none of these features. Instead the cells more or less (depending on species) coalesce into a compact layer which becomes attenuated in the hinge regions. A 30 Å filament pattern can be seen.The mesos layer of squamates resembles the hinge region of crocodilians, exhibiting a combination of the characteristics of both and keratin.This study constitutes publication No. 464 from the Oregon Regional Primate Research Center, supported in part by NIH Grant No. FR-00163.     

Inhibition of DNA polymerase α by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

The impact of the C-terminal domain on the interaction of human DNA topoisomerase II α and β with DNA

Background

Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.

Methodology/Principal Findings

We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.

Conclusions/Significance

The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme''s K_D for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.     

Analysis of chlorophyll fluorescence induction kinetics exhibited by DCMU-inhibited thylakoids and the origin of α and β centres

Analyses of room-temperature chlorophyll fluorescence curves from DCMU-inhibited thylakoids were used to investigate the proposed PS II structural heterogeneity of α and β centres. The kinetics of the area growth curves, representative of Q_A photoreduction, could be modified in the presence of DCMU by exogenous electron acceptors and by added reductants of the PQ pool. The effect of altered DCMU levels (range 0.2–100 μM) on the induction curve kinetics was to modify preferentially the slow-β component, while having only a very small effect on the total variable fluorescence yield. Over the DCMU concentration range used, the unnormalized area of the induction curve (A_max) decreased with increasing herbicide concentration by approx. 45%, indicating that less quanta were required to reduce Q_A. It was found that the dark reoxidation of Q_A in the presence of DCMU and Ant 2p after a light pretreatment regenerated the slow kinetic component. When chlorophyll fluorescence emission at 685 and 731 nm was measured, no difference was observed in the kinetics of the induction curve. The analysis of PS II-enriched, oxygen-evolving membranes indicated the presence of both the fast and slow kinetic components, although this type of preparation showed a modified fast phase. The above observations led to the conclusion that several of the previously proposed characteristics of PS II_α and PS II_β centres do not hold and that a type of PS II heterogeneity involving different degrees of DCMU inhibition is sufficient to explain many of the observations made.