Pancreatic Deoxyribonuclease Research Articles

The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin.

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.

Effect of Mg 2+ and Mn 2+ on hydrolysis of calf thymus DNA by pancreatic deoxyribonuclease I

Divalent cations are activators for DNA hydrolysis by pancreatic deoxyribonuclease I. Apparent V m and K m changes have been studied in presence of Mn 2+ or Mg 2+. The activation process modifies both V m and K m , their relationship with Mg 2+ or Mn 2+ being a linear one. Deoxyribonucleotides inhibit the DNA hydrolysis, whether Mg 2+ or Mn 2+ is the activator; the purine deoxyribonucleotides are more effective as inhibitors than the pyrimidine ones. The effect of some derivatives of adenine has been studied: the inhibition is maximum with 5′-dAMP and minimum with adenine or adenosine. A kinetic mechanism of enzymatic activation by Mn 2+ or Mg 2+ is discussed.

Conductimetric study of the binding of Mn 2+ to bovine pancreatic deoxyribonuclease

The binding of Mn 2+ on bovine pancreatic deoxyribonuclease has been studied by a conductimetric method. At low ionic strengths, a high-affinity single binding site is demonstrated. The association constant value ( K = 1.2 × 10 5 M −1 at pH 8) is high enough to conclude that, in standard experimental conditions for DNA hydrolysis, the reacting species is the DNAase-cation complex. Competitive binding studies in presence of Mg ++ and Ca ++ show that these cations do not bind on the Mn ++ site.

Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage.

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.

Isolation and characterization of multiple forms of ovine pancreatic deoxyribonuclease. Chromatograhpic behavior of the enzyme on concanavalin A-agarose and carboxymethylcellulose columns.

A new procedure has been devised for the purification of ovine DNase, including (NH/4)2SO4 fractionation, two steps of CM-cellulose chromatography, concanavalin A-agarose chromatography, and gel filtration on Sephadex G--100. The enzyme, like bovine DNase, exhibits multiplicity due to changes in the primary structure and the sugar structure of the carbohydrate moiety. Unlike bovine DNase, ovine DNase does not have sialic acid in any of its multiple forms. Concanavalin A-agarose is useful in the purification of not only ovine but also bovine DNase. For ovine DNase, it is a necessary and key step of purification; for bovine DNase, it can be used to purify commercial preparations of DNase free from proteases in a single step as judged by its stability in Ca2+-free media at pH 8.0. The purified enzyme has a specific activity equal to that of a highly purified DNase and presumably contains predominantly DNases A and C. Two of the four forms of ovine DNase have been purified to apparent homogeneity and subjected to chemical analysis. The present results show that bovine and ovine DNases have indistinguishable molecular weights and identical end groups, suggesting that they may have the same number of amino acid residues. The amino acid composition indicates that two enzymes may have six residues of amino acids subject to substitution which can be explained by single base changes in their genetic code words. Amino acid analyses also indicate that the most likely difference between two forms of ovine DNase is the substitution of Leu for Arg.

The variation with age of the structure of chromatin in three cell types from rat liver.

The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.

Preparation of triple-block DNA polymers using recombinant DNA techniques.

The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described. The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase. The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E. coli. Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28. Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site. The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts. Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally.

5'-Nucleotidase reverses the inhibitory action of actin on pancreatic deoxyribonuclease I

digitalData = {"site":{"ip":"31.40.225.101","environment":"LIVE","website":"febs.onlinelibrary.wiley.com","websiteCode":"febs","serverDate":"2022-10-22"},"identities":[{"type":"SmartGroupUser","uuid":"b238ab85-6bb4-49e4-84ca-a0d9f3ee5a35"},{"type":"SmartGroupUser","uuid":"eabce89b-65c2-4a09-ab7c-bd2b3fea2f10"},{"type":"RegionUser","uuid":"3c5060a1-6620-438f-862b-902a4d4cd4ac"},{"type":"RegionUser","uuid":"86f8379b-60ef-449d-b10a-a547ff2452af"},{"type":"SmartGroupUser","uuid":"a86d27d2-6bcd-4b94-bca5-fe16f4882855"},{"type":"SmartGroupUser","uuid":"da88d808-3f7b-4bde-9210-10862b6d121b"},{"type":"SmartGroupUser","uuid":"8c72a733-117a-4bc9-a682-13475f13e6a3"},{"type":"SmartGroupUser","uuid":"756fc162-8722-461f-a077-ebffec4f4fae"},{"type":"SmartGroupUser","uuid":"cf9c85b9-7f3b-4309-aeaa-c41669cabe3b"},{"type":"SmartGroupUser","uuid":"db8b8d10-1aed-4809-b3ee-70938c569014"},{"type":"SmartGroupUser","uuid":"cdce41c2-a8de-4a64-a4d9-a85caaaa2314"},{"type":"SmartGroupUser","uuid":"7a2a316d-5a37-4ccd-afdb-737bfb863a48"},{"type":"SmartGroupUser","uuid":"39f911ce-44b6-4dba-9978-8b2e947a8eb0"},{"type":"SmartGroupUser","uuid":"97587ffd-dcb4-4cb5-9986-a8ce611532bd"},{"type":"SmartGroupUser","uuid":"a46a154d-979f-41ba-b459-fd34df5c071f"},{"type":"SmartGroupUser","uuid":"b7acda8c-ce18-451b-a68d-e14b72f9b5b8"},{"type":"SmartGroupUser","uuid":"055a2bdd-861f-4fa7-bd49-9773db7e0e3e"},{"type":"SmartGroupUser","uuid":"b2dbfe27-ecae-4bb6-bb0d-d75e5caa3762"},{"type":"SmartGroupUser","uuid":"1a6297fb-6679-4882-a4c2-fdd124624edf"},{"type":"SmartGroupUser","uuid":"8032f17e-5050-4fb4-9c40-e1f6875d5c2b"},{"type":"SmartGroupUser","uuid":"ab743440-628f-4964-9216-9983ad3beadf"},{"type":"SmartGroupUser","uuid":"a4187e00-f28e-4754-9a0f-1eb306fb0679"}],"publication":{"item":{"title":"5'‐Nucleotidase reverses the inhibitory action of actin on pancreatic deoxyribonuclease I","doi":"10.1016/0014-5793(78)81012-6","access":"free","earliestDate":"1978-11-15","formats":["Cited by","First page image","PDF","References"],"topics":[{"taxonomyUri":"global-subject-codes","topicLabel":"LIFE SCIENCES","topicUri":"ls"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry","topicUri":"ls36"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry (Chemical Biology)","topicUri":"chb0"},{"taxonomyUri":"global-subject-codes","topicLabel":"CHEMISTRY","topicUri":"ch"},{"taxonomyUri":"global-subject-codes","topicLabel":"Cell & Molecular Biology","topicUri":"ls30"},{"taxonomyUri":"a6ce160f-ca8e-4750-b882-825dad618869","topicLabel":"Research Article","topicUri":"2ef047a8-f5b9-4a45-898a-38d0466e94d9"}]},"group":{"title":"","doi":"10.1002/feb2.1978.95.issue-2","access":"free","volume":"95","issue":"2","coverDate":"1978-11-15","topics":[{"taxonomyUri":"global-subject-codes","topicLabel":"LIFE SCIENCES","topicUri":"ls"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry","topicUri":"ls36"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry (Chemical Biology)","topicUri":"chb0"},{"taxonomyUri":"global-subject-codes","topicLabel":"CHEMISTRY","topicUri":"ch"},{"taxonomyUri":"global-subject-codes","topicLabel":"Cell & Molecular Biology","topicUri":"ls30"}]},"series":{"title":"FEBS Letters","doi":"10.1002/(ISSN)1873-3468","access":"no","code":"18733468","issn":"0014-5793","eissn":"1873-3468","topics":[{"taxonomyUri":"gpt-sitename","topicLabel":"wly.chemistry_000033","topicUri":"wly.chemistry_000033"},{"taxonomyUri":"global-subject-codes","topicLabel":"LIFE SCIENCES","topicUri":"ls"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry","topicUri":"ls36"},{"taxonomyUri":"global-subject-codes","topicLabel":"Biochemistry (Chemical Biology)","topicUri":"chb0"},{"taxonomyUri":"global-subject-codes","topicLabel":"CHEMISTRY","topicUri":"ch"},{"taxonomyUri":"global-subject-codes","topicLabel":"Cell & Molecular Biology","topicUri":"ls30"},{"taxonomyUri":"gpt-zonename","topicLabel":"febs-letters_FEB2","topicUri":"febs-letters_feb2"}]}},"page":{"subPage":"Abstract","page":"Article/Chapter View","pageGroup":"Publication Pages","title":"title","errorCode":200}}

Induction and repair of strand breaks and 3′-hydroxy terminals in the DNA of mouse brain following gamma irradiation

DNA was isolated from mouse brain after in vivo γ-ray irradiation, treated with endonuclease S 1 from Aspergillus oryzae if necessary, and analysed further by alkaline and neutral sucrose gradient centrifugation. In parallel, its template activity was determined by DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay as described previously. Similar experiments were performed with cultured mouse leukaemia cells (L5178Y) irradiated in vitro at 0°C. Irradiation induced single- and double-strand breaks in the DNA of the brain with a yield of 1.0 and 0.1 break per 10 12 dalton per rad (100 eV/break and 770 eV/break), respectively. The yield of single-strand breaks in the brain was lower than that found in the cultured cells, whereas the yield of double-strand breaks was found to be almost the same in both cases. Treatment of irradiated DNA with single-strand-specific S 1 endonuclease gave rise to further breaks detected on neutral sucrose gradient analysis. The yield of these breaks was also higher in the brain compared to the cultured cells. The increase per unit dose in the template activity of the DNA from the brain was found to be five times as much as that found in the cultured cells. Then, the average number of deoxyribonucleotides incorporated per break was determined on DNA which had experienced different treatments. The value for the brain DNA irradiated in vivo was found to be five times as much as that found for DNA treated with pancreatic deoxyribonuclease and 10 times as much as those found for DNA from the cultured cells and isolated brain nuclei irradiated in vitro at 0°C. Thus, in vivo irradiation seemed to induce gaps with 3′-OH terminals in addition to simple breaks with or without 3′-OH terminals found in the cultured cells. Radiation-induced single-strand breaks and 3′-OH terminals in the DNA of the brain were repaired following irradiation. Approx. 20–40% of the terminals or breaks induced were, however, remaining at 3 h or more after irradiation, depending on the dose administered.

Preparation of protease-free and ribonuclease-free pancreatic deoxyribonuclease.

When pancreatic DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, freedom from contaminating ribonucleases or proteases is an important property of the enzyme preparation. A simple one-step procedure has been developed to effect complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose (a column (0.9 X 60 cm) operated in series with a regeneratable 1-ml bed). Commercial preparations of DNase (about 10 mg) give a quantitative yield of the enzyme that is protease-free as evidenced by full stability for more than 10 days at pH 8 and 37 degrees C even in the absence of the protecting action of Ca2+. Removal of the last traces of RNase has been accomplished by affinity chromatography on a column (0.4 X 72 cm) of 5'-(4-aminophenyl-phosphoryl)-uridine 2'(3')-phosphate-Sepharose; the product is a highly active DNase that gives no detectable hydrolysis of RNA by assay on radioactive substrates.

Circular dichroism studies on the effects of ethanol on the conformation of α1-acid glycoprotein, α1-antitrypsin, deoxyribonuclease, pepsinogen, soybean trypsin inhibitor and unfolded ribonucleases

The effects of 25 to 75 volume-% ethanol on conformation of human serum α 1-acid glycoprotein, human serum α 1-antitrypsin, pancreatic deoxyribonuclease I, porcine pepsinogen, the “Kunitz” trypsin inhibitor from soybeans, and oxidized as well as reduced and S-carboxymethylated ribonucleases were tested by the circular dichroism (CD) probe. It was found that 25 volume-% ethanol had a slight effect, whereas 50–75 vol.-% alcohol significantly altered the conformation. The tertiary structure was perturbed and the polypeptide main chain was reorganized into new conformations of higher helix and β-structure contents than in the native state. Comparison of the various proteins showed that the degree of reorganization depended chiefly on the cross-linking of the main chain by disulfide bridges. While the unfolded ribonucleases were refolded by 25 vol.-% ethanol into ordered conformations, the native ribonuclease was resistant even to 50–66% alcohol. The conformation of deoxyribonuclease and α 1-antitrypsin was more sensitive to 25 vol.-% ethanol than the conformation of α 1-acid glycoprotein, pepsinogen, and soybean trypsin inhibitor. Almost complete restoration of the native conformation was achieved by diluting the alcohol-containing solutions with water or by dialysis against water or buffer solutions. However, the renaturation depended on the time of contact with alcohol and on the temperature at which the alcohol-containing solutions were kept.

Isolation and physical studies of the intact supercoiled

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.

The role of Ca2+ on pH-induced hydrodynamic changes of bovine pancreatic deoxyribonuclease A.

DNase A studied by gel filtration on Sephadex G-100 at pH 7.4 in 40 mM Tris-HCl buffer, behaves hydrodynamically as a spherical monomeric macromolecule of around 31,000 molecular weight, with a Stokes radius = 24.7 A, f/fo = 1.19, and D20,W = 8.69. Similar results were obtained by analytical dialysis using zinc chloride-modified cellophane membranes. The elution volume of DNase A decreases as the pH increases between pH 4.7 and pH 9.5. This effect has been attributed to a change in the tridimensional structure of the protein and interpreted as a modification in the axial ratio due to unfolding of the polypeptide chain with increase in the apparent Stokes radius. The addition of Ca2+ produce reversion of the pH-induced changes at pH 9.5. The transition occurs when Ca2+ binds to at least two binding sites (n = 1.66 in a Hill plot) with a Kd = 8.9 X 10(-5) M and the effect appears to be cooperative. These findings support the hypothesis that Ca2+-binding to DNase A causes a conformational change that maintains a more active structure of the enzyme, especially when the pH-induced unfolding reduces its activity.

Enzymatic Digestion of DNA γ-irradiated in Aqueous Solution Separation of the Digests by Ion-exchange Chromatography

Aqueous solutions of DNA were gamma-irradiated in the presence and absence of oxygen and enzymatically hydrolysed by the combined action of pancreatic deoxyribonuclease (DNase I), snake-venom phosphodiesterase (PDE I), spleen phosphodiesterase (PDE II) and alkaline phosphatase. In contrast to unirradiated DNA, which is fully hydrolysed to nucleosides by these enzymes, gamma-irradiated DNA yields a series of oligonucleotides. Their isolation might enalbe the future identification of the chemical nature of DNA lesions.

Purification and Properties of Human Pancreatic Deoxyribonuclease I1

Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.

Ethidium bromide (EB) binding to nucleosomal DNA: Effects on DNA cleavage patterns

Binding of ethidium bromide (EB) to chromatin DNA induces structural changes in nucleosomes. The characteristic cleavage patterns of nucleosomal DNA after digestion with either micrococcal nuclease or pancreatic deoxyribonuclease are altered in the presence of the intercalating dye. Instead, apparently random digestion occurs. Polylysine reduces the amount of EB-binding sites in nucleosomal DNA. Since the intercalation of EB is known to proceed from the minor groove of DNA, polylysine supposedly occupies the same site of the nucleosomal DNA moiety.

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases.

Abstract The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded. The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.

Release of Escherichia coli DNA from membrane complexes by single-strand endonucleases.

Treatment of gently prepared lysates of Escherichia coli with single-strand-specific endonuclease (SI or from mung beans) results in the release of about 90% of the DNA from membranes, as determined by the M band technique. The released DNA has an average molecular weight of about 1.2 X 10(8). Data obtained with endonuclease S1 fit a mathematical model in which substrate sites are at or near membrane attachment sites. Data obtained with pancreatic deoxyribonuclease or x-rays fit a model for double-strand breaks at random sites along the DNA. Fitting data to these models, we estimate that there are 18+/-5 membrane attachment sites. The DNA remaining after S1 nuclease treatment is enriched for the region near the origin of chromosome replication. Therefore, attachment at this region near the origin of chromosome replication. Therefore, attachment at this region appears to be chemically different from that at the other sites along the DNA.

The isolation of a high-molecular-weight glycoprotein from pig colonic mucus.

The principal components of a variety of mucous secretions have been found to be glycoproteins, many of mol.wt. 500000 or more (Gottschalk, 1972). We have isolated the glycoprotein from pig colonic mucus and examined its structure in order to compare it with that of the well-characterized glycoprotein of the gastric mucus from the same species (Snary & Allen, 1971 ; Allen, 1977). Mucosal scrapings were prepared from the washed colons of freshly slaughtered pigs. After exhaustive dialysis of the scrapings against water containing 0.02 % azide, the water-soluble mucus was separated from the insoluble mucous gel and cell debris by squeezing through muslin followed by centrifugation (Fig. 1). The water-soluble mucus, which represented 70% dry wt. of the non-diffusible mucus, contained 72% by wt. protein, 10% by wt. nucleic acid and 4.6% by wt. glycoprotein. Nucleic acid was completely removed by digestion of the water-soluble mucus (concentration 4-7 mg/ml) with proteinase-free bovine pancreatic ribonuclease (EC 3.1.4.22; final concentration 15 pg/ml) and ox pancreatic deoxyribonuclease (EC 3.1.4.5; final concentration 2.5,ug/ml). Incubation was for 17 hat 37C, after which all the nucleic acid was diffusible. The non-covalently bound protein was separated from the glycoprotein by equilibrium density-gradient centrifugation in CsCl (Starkey et al., 1974). Owing to the high ratio of non-covalently bound protein to glycoprotein, it was necessary to repeat this step (on the recovered glycoprotein) in order to achieve complete separation of the two (Fig. 1). The purified glycoprotein was subsequently fractionated into a high-molecularweight glycoprotein A (excluded) and a low-molecular-weight glycoprotein fraction B (included) by gel filtration on Sepharose 4B. The high-molecular-weight glycoprotein A was the principal component, accounting for 84 % of the glycoprotein recovered from the Sepharose column. The low-molecular-weight minor component, the glycoprotein fraction B, has a similar ratio of sugars to glycoprotein A (Table 1) but a higher protein content (40 % by wt.). The high-molecular-weight glycoprotein A had a sugar analysis characteristic of glycoproteins from other mucous secretions (Gottschalk, 1972) with both sulphate and sialic acid present (Table 1). The glycoprotein possessed A blood-group activity (1.25,ug/ 0.lml; assayed by the method of Kabat & Bezer, 1945) which was doubled (0.63,ug/ 0.1 ml) by removal of the sialic acid residues with neuraminidase. A full complement of amino acids was present, although threonine, serine and proline comprised 54 % of the total residues. It is noteworthy that the blood-group activity and both the sugar and the amino acid composition of the colonic glycoprotein are similar to that of the glycoprotein from pig gastric mucus (Starkey et al., 1974), except that the colonic glycoprotein has more sialic acid and less fucose and galactose. Further similarities between the glycoproteins from the two mucous secretions were apparent after Pronase digestion. The Pronase-digested glycoprotein from colonic mucus was separated from the peptides and added Pronase by equilibrium density-gradient centrifugation in CsCl. Analysis of the Pronase-digested glycoprotein showed that carbohydrate had been conserved, but 29.8% of the peptide was lost. Threonine, serine and proline comprised 75% of the remaining amino acids, showing a proportionately bigger loss in the other amino acids. This results points to the presence of non-glycosylated and glycosylated regions of the peptide core in the colonic glycoprotein similar to that found for glycoproteins from other mucous secretions (Donald, 1973 ; Scawen &Allen, 1975). The presence of sialic acid and sulphate together in the same glycoprotein has been shown for other glycoproteins from mucous secretions, for example sheep colonic mucus (Kent & Marsden, 1963) and pig gastric mucus (Starkey et al., 1974).

Enzymic repair of alkylated DNA in vitro

The damage-directed strand incision step in the nucleotidyl DNA excision-repair pathway (NDERP) was characterized in quiescent monolayer cultures of human fibroblasts in which the plasma membrane was selectively permeabilized with saponin. When permeable normal human fibroblasts (NHF) were incubated in a DNA-repair assay mixture lacking the deoxyribonucleoside triphosphate precursors, the numbers of UV-dependent DNA-strand breaks were increased by about 9-fold consistent with the uncoupling of incision from gap-filling DNA synthesis and ligation. In uncoupled NHF omission of ATP reduced the numbers of UV-dependent strand breaks by 84% confirming the requirement for ATP for reparative strand incision. Time-course experiments indicated that the maximum rate of strand incision occurred in the first 10 min of incubation of permeable cells and diminished to 16–28% of this rate between 30 and 60 min of incubation. The initial rate of incision in permeable NHF was estimated to be 20% of that seen in intact fibroblasts. Dose-response studies indicated an initial saturation of strand incision activity at fluences between 10 and 25 J/m2. In permeable group A xeroderma pigmentosum fibroblasts (XPA) few UV-dependent incisions were produced after 10–25 J/m2. In the xeroderma pigmentosum variant (XPV) strain that we studied, strand incisions saturated at a plateau level that was about twice that seen in the NHF strain suggesting the preservation of a higher level of incision activity after permeabilization. After fluences above 50 J/m2 additional strand incision was observed in all cell strains reflecting the activity of a damage-dependent endodeoxyribonuclease that is independent of the NDERP. Saponin-treated fibroblasts were also permeable to pancreatic deoxyribonuclease I and the UV-DNA endonuclease from M. luteus indicating that these preparations may be used for in vitro complementation.