De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.     

Onset of deoxyribonucleic Acid synthesis in germinating wheat embryos

Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos.     

Transient expression and activity of human DNA polymerase iota in loach embryos

Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.     

DNA-dependent RNA polymerase activities during embryogenesis in the bug, Oncopeltus fasciatus

Using α-amanitin to inhibit polymerase II activity in intact nuclei from Oncopeltus embryos, it is demonstrated that there is no difference in relative amounts of α-amanitin-resistant (Form I) and α-amanitin-sensitive (Form II) polymerases at two stages of embryonic development (70 and 140 hr), although the total polymerase activity is considerably higher at the earlier stage. However the RNA made under these circumstances (presumably due to Form I activity) appears to be, as expected, largely ribosomal.When the RNA polymerase activities are solubilized and separated, there is a substantially higher level of Form I activity in 70-hr embryos over that in 140-hr embryos. It is suggested that this high level of polymerase activity is correlated directly with the high level of ribosomal RNA synthesis at this stage.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

A DNA polymerase alpha partially resistant to aphidicolin in cells and embryos of Med-fly (Ceratitis capitata Wied.)

We have studied the DNA polymerase activities in cultured cells and embryos of Med-fly (Ceratitis capitata Wied.) and we observed that only DNA polymerases alpha and gamma were detectable in crude extracts. The level of DNA polymerase alpha, measured during the life cycle of the insect embryos, increased in parallel with the rate of embryonic cell proliferation, whereas DNA polymerase gamma increased at much later fertilization time, when cell differentiation is already taking place. DNA polymerase alpha, purified 100 folds from Med-fly embryos, was 10 times more resistant to aphidicolin, its specific inhibitor, than the mammalian DNA polymerase alpha. In situ visualization of the active peptides after NaDodSO4/PAGE, confirmed that only high Mr DNA polymerase fragments were present in embryo extracts and in purified preparations of DNA polymerase alpha. It appears that C. capitata cells represent a rather peculiar system in the phylogeny of DNA polymerases since they are devoid of DNA polymerase beta and present a DNA polymerase alpha partially resistant to aphidicolin.     

Effect of spermine on interaction of DNA polymerase α from the loach (Misgurnus fossilis) eggs with DNA

Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase α on activated (gapped) DNA. The stimulatory effect increases in the order: putrescine, spermidine, spermine. Kinetic analysis shows that spermine does not change the affinity of the polymerase for dTTP, but it decreases the enzyme affinity for DNA. The apparent K_m of the polymerase for activated DNA progressively increases from 14 to 1200 μM (nucleotide), if the concentration of spermine rises up to 2 mM, while V_max reaches a maximum at 0.5 mM spermine and then drops at higher polyamine concentrations. Native calf thymus DNA and especially single-stranded DNA from phage M13 appear to be inhibitors of α-polymerase activity on gapped DNA. Dixon plots suggest simple competitive inhibition of the polymerase activity by single- or double-stranded DNA and absence of cooperativity in the interaction of the polymerase with DNA. Hill-plot analysis is compatible with the interpretation that there is only one DNA binding site on each DNA polymerase α molecule. Spermine, even at low concentrations, decreases sharply the affinity of the enzyme for double-stranded DNA, while the enzyme affinity for single-stranded DNA changes insignificantly. Another result of spermine action is the destabilization of the polymerase-DNA complex. The ratio of the ‘static affinity’ of the enzyme to its ‘kinetic affinity’ decreases 2.2-fold in the presence of 0.5 mM spermine. As a result, the sensitivity of DNA synthesis to 3′-deoxy-3′-aminothymidine 5′-triphosphate and to 1-β-d-arabinofuranosylcytidine 5′-triphosphate decreases in the presence of the polyamine. Both spermine effects, the decrease in the ‘nonproductive binding’ of the polymerase to double-stranded regions in DNA and the destabilization of the polymerase-DNA complex, presumably account for the increase in the activity of the loach α-polymerase on activated DNA.     

Comparison of DNA Polymerase of Rhizobium meliloti and Alfalfa Bacteroids

DNA dependent-DNA polymerase activity was established and partially purified from extracts of cultured Rhizobium meliloti, F-28, and nodule bacteroids (R. meliloti, F-28) of alfalfa plants (Medicago sativa). Polymerase activity in the partially purified fractions showed characteristic dependence on Mg²⁺, DNA, and a full complement of deoxyribonucleoside triphosphates. DNase activity, preference of “activated” double strand DNA, and inhibition by p-chloromercuribenzoate and MnCl₂ were responses common to both systems. The two systems however did exhibit some differences in pH, Mg²⁺, and primer optima. Polymerase activity in crude extracts of the cultured bacteria was more stable and had 10- to 18-fold greater specific activity than the bacteroid extracts. Preliminary measurements of specific DNA polymerase activity in crude extracts of cultured Rhizobium japonicum were not significantly higher than that in the crude extracts of soybean nodule bacteroids. A possible correlation between DNA synthesis and the successful establishment of rhizobia-legume symbiosis is discussed.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

Endonuclease V-mediated deoxyinosine excision repair in vitro

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi⁺ E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3′-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.     

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia

Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues (Golosow and Grobstein, 1962), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al., 1964). We have shown that this fraction also stimulates the incorporation of thymidine-³H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen.The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine-³H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine-³H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly.MF also affected RNA and protein synthesis. The rate of leucine-³H incorporation into protein and uridine-¹⁴C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.     

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and uv-induced mutagenesis.

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol^?) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol^? mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol⁺ parent. The presence of FP18 or FP111 in the Pol^? mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant.The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla.The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.     

Denervation effects on newt limb regeneration: DNA polymerase activity in vitro

DNA polymerase activity in extracts of newt (Triturus viridescens) tissues was assayed in an effort to characterize the effect of nerve resection on the regeneration of amputated limbs. Regenerating limbs display 5–20 times more polymerase activity than nonregenerating limbs. Denervation of partially regenerated limbs, which completely prevents further regeneration in vivo does not, however, affect DNA polymerase activity assayed in vitro. These results suggest that while DNA polymerase activity is responsive to the stimulus of regeneration, denervation effects are probably not directly exerted on this enzyme activity.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.     

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.     

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l^-/- Polh^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l^-/- Polh^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.