Developmental changes of DNA ligase during ram spermatogenesis

The molecular forms and activities of ram DNA ligase have been investigated during spermatogenesis from the stage of early round spermatids to ejaculated spermatozoa. Through germ cell maturation, two consecutive forms of the enzyme (6S and 7S) have been found. The 6S form (DNA ligase II) is observed in primary and secondary spermatocyte, as well as in round spermatids. The 7S form (DNA ligase I) is present in elongated spermatids and in the sole round cell population with spermatogonia and young primary spermatocytes. In ram germ cells, DNA ligase I and DNA ligase II appear to be respectively associated with DNA replication repair. The absence of DNA ligase II associated with the absence of DNA repair in testicular and ejaculated spermatozoa might be related to male infertility.     

Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells

Three endonucleases from murine plasmacytoma cells that specifically nick DNA which was heavily irradiated with ultraviolet (UV) light were resolved by Sephacryl S-200 column chromatography. Two of these, UV endonucleases I and II, were purified extensively. UV endonuclease I appears to be a monomeric protein with a molecular mass of 43 kDa; UV endonuclease II has an S value of 2.9 S, with a corresponding molecular mass estimated at 28 kDa. Both enzymes act as a class I AP endonuclease, cleaving phosphodiester bonds via a beta-elimination mechanism, so as to form an unsaturated deoxyribose at the 3' terminus. Both have thymine glycol DNA glycosylase activity and their substrate specificities generally appear to be overlapping but not identical. UV endonuclease I acts on both supercoiled and relaxed DNAs, whereas II acts only on supercoiled DNA. Both enzymes are active in EDTA, but have different optima for salt, pH, and Triton X-100. Each enzyme is also present in cultured diploid human fibroblasts.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

Properties of acid beta-N-acetyl-D-glucosaminidase from cock semen.

Two forms (I and II) of beta-N-acetyl-D-glucosaminidase from cock seminal plasma and one form (III) from spermatozoa were separated by chromatofocusing. The active enzyme forms I and II had pI values of 6.6 and 6.3, respectively, while form III had two subforms with pI values of 6.3 and 6.1, as determined by polyacrylamide gel electrofocusing. The molecular weights were 76,000 for forms I and III and 32,000 for form II. The optimum pH of enzyme forms I and III ranged from 3.6 to 4.0. In contrast, form II showed one distinct maximum at pH 3.7. The Km values obtained with p-nitrophenyl-beta-N-acetyl-D-glucosaminide as substrate were 0.35, 0.28, and 0.39 mM for forms I, II, and III, respectively. It is assumed that both cock spermatozoa and cock seminal plasma contain a common, enzymatically active beta-N-acetyl-D-glucosaminidase subunit with M(r) about 32,000 and pI 6.3.     

Identification of two activities of (ADP-ribose)n glycohydrolase in HeLa S3 cells

HeLa S3 cells contained two activities (form I and II) that degrade (ADP-ribose)n exo-glycosidically. Form I was extracted from nuclei only by sonication in high ionic strength, while form II was soluble in cytosol. The two active forms differed in chromatographic behaviors, in their Km values for (ADP-ribose)n, and in their pH and salt requirements for optimal activity, although both forms exhibited properties characteristic of (ADP-ribose)n glycohydrolase such as requirement of sulfhydryl compounds and sensitivity of ADP-ribose and cAMP. Form I and II had apparent molecular weights of 72,000 and 53,000, respectively, as determined by gel filtration on Sepharose CL-6B.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II

DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

Studies on DNA polymerases and topoisomerases in archaebacteria

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.     

Three deoxyribonucleic acid-dependent adenosine triphosphatases from Bacillus subtilis.

We have isolated from Bacillus subtilis three deoxyribonucleic acid (DNA)-dependent adenosine triphosphatases (ATPases) (gamma-phosphohydrolases). The enzymes were extensively purified, and their physicochemical and functional properties were determined. The three enzymes (ATPases I, II, and III) were shown to be different by several criteria. ATPases II and III showed an absolute requirement for single-stranded DNA as a cofactor, whereas ATPase I had some residual activity also with double-stranded DNA. They required Mg2+ and had a pH optimum of 6.5 to 7. Only adenosine 5'-triphosphate and deoxyadenosine 5'-triphosphate were hydrolyzed. The molecular weights of ATPases I, II, and III were 108,000, 115,000, and 148,000, respectively. Km values for adenosine 5'-triphosphate and DNA were also evaluated and shown to be different for each enzyme. All three enzymes formed physical complexes with single-stranded DNA. We present evidence that ATPases I and II might migrate along DNA during adenosine 5'-triphosphate hydrolysis. On the other hand, this effect was not observed with ATPase III, which exhibited the highest affinity for single-stranded DNA.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

Anatomy of Bovine Mammillitis DNA I. Restriction Endonuclease Maps of Four Populations of Molecules That Differ in the Relative Orientation of Their Long and Short Components

In this paper, we report that the DNA of bovine mammillitis virus (BMV) consists of two covalently linked components that are 71.5 x 10(6) and 15.7 x 10(6) in molecular weight and designated L and S, respectively. We further report that the BMV DNA consists of four equimolar populations differing only in the orientation of the L and S components relative to each other. This conclusion is based on the following: (i) The sum molecular weight of fragments generated by digestion of BMV DNA with Hsu I, Hpa I, Bgl II, or Xba I significantly exceeds the established molecular weight of the intact DNA. (ii) In each digest, the fragments form three groups differing in molar concentration. In reference to the molar concentration of intact DNA, each enzyme digest contained a set of four fragments 0.25 M in concentration, a set of four fragments 0.5 M in concentration, and a variable size set, unique for each enzyme digest, 1.0 M in concentration. (iii) Experiments involving digestion of intact DNA by lambda exonuclease followed by restriction endonuclease digestion established that each of four 0.5 M fragments were positioned at the termini of the BMV DNA. (iv) Complete maps for the fragments generated by each enzyme established that the 0.25 M fragments arise by fusion of the sequences of the terminal fragments when these are juxtaposed as a consequence of the inversion of L and S components. The maps also established the dimensions of the L and S components. We conclude that the structure of BMV DNA is similar to that of HSV DNA previously shown to consist of two unequal size components that invert relative to each other.     

Characterization of three enzymatic forms of glucose-6-phosphate dehydrogenase from Aspergillus oryzae

Three forms (I, II and III) of glucose-6-phosphate dehydrogenase were isolated from mycelium of Aspergillus oryzae grown on ribose as the carbon source, by ion-exchange chromatography. The Km values determined for the three forms with respect to glucose-6-phosphate were nearly identical; however the Km for NADP+ were different and the Vmax for the isoenzymatic form II was higher than those for I and III. Inhibition by NADPH was competitive with respect to NADP+, isoenzyme II showing the highest Ki. The optimum pH for forms I, II and III were 9.0, 8.0 and 8.5, respectively, and form I was more thermostable than the others. The apparent molecular weights, determined by gel filtration, were 92,000, 117,500 and 141,000 for forms I, II and III, respectively.     

Nuclear DNA polymerases and the HeLa cell cycle.

Purified nuclei of HeLa S3 cells contain two DNA-dependent DNA polymerases that have distinct physical and enzymatic properties. We have investigated the variations in their activity during the cell cycle of a synchronized culture. Cells were synchronized by a double thymidine block, harvested at various phases of the cycle, and the two DNA polymerases were purified partially by DEAE-cellulose and phosphocellulose chromatography. The activity of DNA polymerase I (low molecular weight, N-ethylmaleimide-insensitive) remains essentially constant throughout the cycle. The activity of DNA polymerase II (high molecular weight, N-ethylmaleimide-sensitive), however, increases during G1 to mid-S and declines, 7- to 10-fold between late-S and G2. Addition of cycloheximide (60 mug/ml) to cultures 12 hours after the release from thymidine block abolishes the rise in the activity of DNA polymerase II. Cycloheximide also reduced the activity of DNA polymerase I by 60%. Addition of hydroxyurea (1mM) at 1 hour after release has no effect on the activity of either enzyme. We conclude that in HeLa cells, DNA polymerase I and II are distinct enzymes, that DNA polymerase II probably functions in DNA replication and is probably induced in response to stimuli for DNA biosynthesis.     

Molecular packing in a second monoclinic crystal of deoxygenated sickle hemoglobin

A close correspondence has been demonstrated between double filaments of deoxygenated hemoglobin S molecules as found in monoclinic crystals, forms I and II, and in sickle fibers. We have carried out a low resolution study of monoclinic form II by X-ray diffraction analysis. Its structure differs from that of form I solely by a shift along the a-axis of the molecular centers of the asymmetric unit, which forms the double filament. The magnitude of the translation was determined from a minimum residual calculation. The x co-ordinates of the symmetry related molecular centers of antipolar double filaments are approximately the same. This means that the double filaments are nearly in register. A minor component associated with form II crystals proved to be form I. The possible existence of additional forms is discussed.The significance of the molecular arrangement in form II is related to its presence in sickle fibers. We have determined the contacts between antipolar double filaments in this form as well as a number in form I not tabulated previously. These new contacts represent additional stabilizing interactions that might provide targets for the design of stereospecific antisickling agents.     

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.     

Characterization of Simian virus 40 DNA component II during viral DNA replication

Replicating molecules of Simian virus 40 DNA labeled during a short pulse with [³H]thymidine have been fractionated by ultracentrifugation methods and the open circular form (DNA component II) has been characterized. The pulse-labeled DNA component II is a relatively small constituent (1 to 3%) of the pool of replicating molecules. Examination of the circular (18 S) and linear (16 S) strands of DNA component II by alkaline sedimentation and by degradation using exonuclease III of Escherichia coli reveals that the newly synthesized DNA is principally in the linear strand. Cleavage of pulse-labeled DNA component II by an fi⁺, R-factor restriction endonuclease from E. coli demonstrates that the interruption in the pulse-labeled strand is specifically located at the termination point for replication.During a chase period of 20 minutes the amount of DNA component II increases to about 6 to 8% of the total labeled viral DNA. The kinetics of formation of superhelical, DNA component I and disappearance of replicative intermediates are linear during the chase period. After several hours of continuous labeling of replicating viral DNA, the DNA component II pool consists mainly of molecules labeled in both strands with the interruption non-specifically located in either strand. These molecules probably arise by the random introduction of single-strand breaks in newly synthesized DNA component I. During short periods of continuous labeling with [³H]thymidine, the ratio of DNA components I to II increases as a function of the pulse duration. These results support a model for 8V 40 DNA replication in which the open circular form is a precursor of the superhelical form.