Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase alpha and beta. Extraction, separation, characterization and changes during spermatogenesis.

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.     

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.     

Lipid peroxidation and the polymerase activities of liver chromatin fractions in rats during aging

Lipids, which enter the composition of actively transcribed and repressed chromatin fractions are found to undergo a peroxidation. This process decreased with aging and more pronounced in actively transcribed chromatin fraction. A decreased activity of DNA polymerase beta and increased activity of RNA polymerase I in this chromatin fraction with aging were observed. It is assumed that observed changes of genome function of old animals may be caused by decreased peroxidation of chromatin lipids.     

Immunocytochemical localization of chick DNA polymerases alpha and beta +

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.     

DNA strand breaks in rat tissues as detected by in situ nick translation

The nick-translation procedure without external addition of DNase was performed in situ on sections of various rat organs to detect possible DNA single-strand breaks (nicks) in normal tissues. The freshly frozen sections were briefly fixed in ethanol/acetone and nick-translated in the presence of E. coli DNA polymerase I. A significant difference in the amount of nuclear reaction was found among the different cell populations as detected by autoradiography following incorporation of tritiated TTP as well as by histochemical staining following incorporation of biotin-dUTP into nuclei. Such incorporation of triphosphates was localized in the DNA and was entirely dependent on E. coli DNA polymerase I. The nuclei with the highest reactivity were found in skeletal muscle cells, lymphocytes in various lymphatic organs, the proliferative cells in the gastrointestinal tract, stratified squamous epithelial cells, duct epithelial cells of salivary gland and the maturing spermatids in the seminiferous tubules. These results suggest that, under the conditions adopted, the cells in various tissues reveal different chromatin structures resulting in varying rates of nick translation reaction. Such difference(s) in chromatin structure, presumably including that in the number of DNA single-strand breaks or in the level of endogenous nuclease activity, may be associated with the mechanisms involved in cell growth and differentiation.     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.     

Effect of the antitumor antibiotic bleomycin on DNA polymerase activity

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.     

A DNA template recognition protein: partial purification from mouse liver and stimulation of DNA polymerase alpha

A protein that specifically enhances up to 13-fold the rate of copying of poly(dT) template by DNA polymerase alpha was partially purified from chromatin of regenerating mouse liver cells. This stimulatory protein, designated herein factor D, also increases 2-3-fold the activity of polymerase alpha with heat-denatured DNA and with primed, circular single-stranded phi X174 DNA. However, factor D has no detectable effect on the copying by polymerase alpha of poly(dG), poly(dA), and poly(dC) templates. Activity of mouse DNA polymerase beta is not affected by factor D with all the tested templates. In contrast to polymerase alpha, factor D is resistant to inactivation by N-ethylmaleimide and calcium ions, but it is readily heat-inactivated at 46 degrees C and is inactivated by trypsin digestion. Partially purified factor D is not associated with detectable activities of DNA polymerase, DNA primase, deoxyribonucleotidyl terminal transferase, and endo- or exodeoxyribonuclease.     

DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).     

DNA polymerase alpha and beta in the California urchin.

DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species.     

Base excision repair in nucleosomes lacking histone tails

Recently, we developed an in vitro system using human uracil DNA glycosylase (UDG), AP endonuclease (APE), DNA polymerase beta (pol beta) and rotationally positioned DNA containing a single uracil associated with a 'designed' nucleosome, to test short-patch base excision repair (BER) in chromatin. We found that UDG and APE carry out their catalytic activities with reduced efficiency on nucleosome substrates, showing a distinction between uracil facing 'out' or 'in' from the histone surface, while DNA polymerase beta (pol beta) is completely inhibited by nucleosome formation. In this report, we tested the inhibition of BER enzymes by the N-terminal 'tails' of core histones that take part in both inter- and intra-nucleosome interactions, and contain sites of post-translational modifications. Histone tails were removed by limited trypsin digestion of 'donor' nucleosome core particles and histone octamers were exchanged onto a nucleosome-positioning DNA sequence containing a single G:U mismatch. The data indicate that UDG and APE activities are not significantly enhanced with tailless nucleosomes, and the distinction between rotational settings of uracil on the histone surface is unaffected. More importantly, the inhibition of pol beta activity is not relieved by removal of the histone tails, even though these tails interact with DNA in the G:U mismatch region. Finally, inclusion of X-ray cross complement group protein 1 (XRCC1) or Werner syndrome protein (WRN) had no effect on the BER reactions. Thus, additional activities may be required in cells for efficient BER of at least some structural domains in chromatin.     

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.