ATP-dependent Deoxyribonuclease Research Articles

Genetic recombination in Neurospora crassa affected by changes in the supply of cellular energy.

HereditasVolume 69, Issue 2 p. 315-319 Open Access Genetic recombination in Neurospora crassa affected by changes in the supply of cellular energy Lars Landner, Corresponding Author Lars LandnerInstitute of Genetics Box 6801 S-11386 Stockholm, SwedenSearch for more papers by this author Lars Landner, Corresponding Author Lars LandnerInstitute of Genetics Box 6801 S-11386 Stockholm, SwedenSearch for more papers by this author First published: December 1971 https://doi.org/10.1111/j.1601-5223.1971.tb02445.xCitations: 4AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Literature cited Abel, W. O. 1964. Untersuchungen über den Einfluss der Temperatur auf die Rekombinationshäufigkeit bei Sphaerocarpus. Z. Vererbungsl. 95: 306– 317. Barbour, S. D. and Clark, A. J. 1970. Biochemical and genetic studies of recombination proficiency in Escherichia coli, I. Enzymatic activity associated with rec B+ and rec C+ genes. Proc. Nat. Acad. Sci. 65: 955– 961. Beers, R. F., Handley, D. D. and Steiner, R. F. 1958. Inhibition and activation of polynucleotide phosphory-lase through the formation of complexes between acridine orange and polynucleotides. Nature 182: 242– 244. Catcheside, D. G. 1968. The control of genetic recombination in Neurospora crassa. — In Replication and recombination of genetic material (Eds. W. J. Peacock and R. D. Brock), Austr. Acad. Sci., Canberra , p. 216– 226. Ephrussi, B. 1953. Nucleo-cytoplasmic relations in microorganisms. — Oxford Univ. Press. New York . Goldmark, P. J. and Linn, S. 1970. An endonuclease activity from Escherichia coli absent from certain rec- strains. Proc. Nat. Acad. Sci. 67: 434– 441. Griffiths, A. J. F. 1968. Heterokaryotic transmission of factors affecting recombination in Neurospora crassa. Can. J. Genet. Cytol. 10: 248– 252. Henderson, S. A. 1970. The time and place of meiotic crossing-over. Ann. Rev. Genet. 4: 295– 324. Kihlman, B. A. 1961a. Biochemical aspects of chromosome breakage. Advan. Genet. 10: 1– 59. Kihlman, B. A. 1961b. Cytological effects of phenylnitrosamines — I. The production of structural chromosome changes in the presence of light and acridine orange. Radiat. Bot. 1: 35– 42. Lamb, B. C. 1969. Evidence from Sordaria that recombination and conversion frequencies are partly determined before meiosis, and for a general model of the control of recombination frequencies. Genetics 63: 807– 820. Landner, L. 1969. Genetic control of recombination. I. Neurospora. Hereditas 63: 460. Landner, L. 1970. Variation of recombination frequency in Neurospora crassa following temperature changes prior to and during meiosis and evidence for a premeiotic sensitive stage. Mol. Gen. Genet. 109: 219– 232. Landner, L. 1971a. Genetic control of recombination in Neurospora crassa: Correlated regulation in unlinked chromosome intervals. Heredity 27:. Landner, L. 1971b. A possible causal relationship between iron deficiency, inhibition of DNA synthesis and reduction of meiotic recombination frequency in Neurospora crassa. Submitt. Mutat. Res.. Lehman, I. R. 1967. Deioxyribonucleases: their relationship to deoxyribonucleic acid synthesis. Ann. Rev. Biochem. 36: 645– 668. Linn, S. and Lehman, I. R. 1965. An endonuclease from Neurospora crassa specific for polynucleotides lacking an ordered structure. J. Biol. Chem. 240: 1287– 1293. Nagai, S., Yangishima, N. and Nagai, H. 1961. Advances in the study of respiration-deficient (RD) mutation in yeast and other microorganisms. Bacteriol. Rev. 25: 404– 426. Oishi, M. 1969. An ATP-dependent deoxyribonuclease from Escherichia coli with a possible role in genetic recombination. Proc. Nat. Acad. Sci. 64: 1292– 1299. Rabin, E. Z., Mustard, M. and Fraser, M. J. 1968. Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa. Can. J. Biochem. Physiol. 46: 1285– 1291. Racker, E. 1965. Mechanisms in bioenergetics. — Acad. Press, New York , London , 375 p. Swanson, R. F. 1971. Incorporation of high molecular weight polynucleotides by isolated mitochondria. Nature 231: 31– 34. Valentin, J. 1971. Recombination in Drosophila melanogaster females is affected by the glycolytic inhibitor, sodium fluoride. Submitt. Mutat. Res.. Westergaard, M. and Mitchell, H. K. 1947. A synthetic medium favouring sexual reproduction. Am. J. Bot. 34: 573– 577. Whitehouse, H. L. K. 1969. Towards an understanding of the mechanism of heredity. — 2. ed., E. Arnold Ltd., London , 447 p. Citing Literature Volume69, Issue2December 1971Pages 315-319 ReferencesRelatedInformation

Kinetic Studies of DNA Cleavage Reactions Catalyzed by an ATP-Dependent Deoxyribonuclease on a 27-MHz Quartz-Crystal Microbalance

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

A spectrophotometric method to quantify linear DNA

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis–Menten kinetics ( K m of 0.6 μM, and a V max of 30 nmol/min/mg). The enzyme requires Mg 2+-ATP and Mg 2+-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 μg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

Direct monitoring of DNA cleavages catalyzed by an ATP-dependent deoxyribonuclease on a 27 MHz quartz-crystal microbalance.

Kinetic studies of enzymatic DNA cleavage reactions (the enzyme binding, hydrolysis along DNA strands, and then release of the enzyme from the completely hydrolyzed ssDNA) were carried out on a 27 MHz quartz-crystal microbalance.

Expression of the ATP‐dependent deoxyribonuclease of Bacillus subtilis is under competence‐mediated control

Transcription of the ATP-dependent deoxynuclease operon (addAB), as monitored by means of an addAB-lacZ transcriptional fusion, has a low, constitutive level and is initiated from a sigma A type promoter. Transcription of addAB is independent of DNA-damaging agents known to induce the SOS response in Bacillus subtilis. However, addAB transcription increased significantly during competence development. This competence-specific induction was dependent on the gene products of srfA, degU and comK, but not on that of recA. Deletion analysis of the addAB promoter region demonstrated that the competence-specific transcription induction requires DNA sequences located upstream of the addAB promoter that associated with ComK, the competence transcription factor. The latter finding indicates that a direct regulatory link exists between the establishment of the competent state and the synthesis of AddAB, required for recombination of internalized donor DNA.

Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits

The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB, RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.

Localization of a Determinant Mediating Partial Macrolide Resistance in Staphylococcus aureus

Four out of more than 8,200 Staphylococcus aureus strains isolated in Japan between 1961 and 1980 were constitutively resistant to a variety of macrolide antibiotics except tylosin and rokitamycin, but susceptible to lincosamide and streptogramin type B antibiotics (PM). The data obtained by agarose gel electrophoresis, CsCl-ethidium bromide density gradient analysis, diagnosis with ATP-dependent deoxyribonuclease, and a test transducing into a rec- mutant with phage 80L2 propagated on PM-resistant S. aureus all suggested that the determinant for the PM-resistance is located in chromosome.

Sequence repetition and genomic distribution of small polydisperse circular DNA purified from HeLa cells

Covalently closed circular DNA molecules (cccDNA) from the human HeLa cell line were purified (96% pure by weight) by use of ATP-dependent deoxyribonuclease, and cloned into the HindIII site of phage λ vector Charon 7. From the cccDNA library thus obtained, nine recombinants carrying mitochondrial DNA and 36 recombinants carrying small polydisperse circular (spc) DNA were picked at random for subsequent tests. The inserted fragments of spcDNA ranged in size from 0.6 to 7.6 kb with a mean length of 1.9 kb, a value which is the same as the average length of spcDNA. Analysis of the cloned spcDNA fragments revealed that (a) all the spcDNA clones investigated shared homologies with chromosomal DNA sequences, (b) all but one cloned DNA contained repetitive sequences, (c) the sequence organization could be roughly classified according to the reiteration frequency as > 10 5 ( Alu family class), 10 4 to 10 5 ( KpnI family class), 10 3 to 10 4 (mitochondrial DNA class) and < 10 3 times per haploid genome, and (d) most of the repetitive sequences were dispersed in the genome, although some appeared clustered.

Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease

Small polydisperse circular (spc) DNAs of mouse thymocytes were purified by a procedure involving nitrocellulose column chromatography and the treatment of ATP-dependent DNase, which acts only upon linear DNA molecules. Nitrocellulose column chromatography prior to the enzyme treatment was essential because digestion of linear DNA duplexes by the enzyme was inhibited by the presence of concomitant single-stranded DNAs. Mitochondrial DNAs were eliminated by linearization with XhoI and digestion with ATP-dependent DNase. The size distribution of the purified spc DNA molecules ranged from 0.2 μm to more than 28 μm, with a mean length of 5.4 μm. Circular molecules of more than 0.4 μm long (or 1.2 kb) were free from the contamination of linear DNA fragments and pure enough to be cloned into plasmids.

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione.

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.

Two pH optima of adenosine 5'-triphosphate dependent deoxyribonuclease from Bacillus laterosporus.

The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.

A lymphocyte ATP-dependent deoxyribonuclease. Isolation and properties.

An ATP-dependent deoxyribonuclease was isolated from lymphocyte nuclei. The enzyme preparation sediments with about 4 S through sucrose gradients and shows one stainable band after sodium dodecyl sulfate gel electrophoresis. We find three, possibly four, activities associated with the enzyme: a DNA-independent ATPase activity; an ATP-independent endonuclease; an ATP-dependent nuclease which degrades nicked DNA to acid-soluble material; and an unwinding activity producing single-stranded regions in nicked DNA.

Purification and some properties of ATP-dependent deoxyribonuclease of Caulobacter crescentus

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and is most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.

Inhibitory Effect of Pyridoxal 5'-Phosphate on the DNA Binding Site of ATP-Dependent Deoxyribonuclease from &lt;italic&gt;Bacillus laterosporus&lt;/italic&gt;

Bacillus laterosporus ATP-dependent deoxyribonuclease has been found to be inhibited by pyridoxal 5'-phosphate. The inhibition is specific for pyridoxal 5'-phosphate and pyridoxal which are required in relatively high concentrations. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine are ineffective. The inhibition is reversed by dilution or dialysis but can be changed to an irreversible inactivation by reduction of the enzyme . pyridoxal 5'-phosphate complex with sodium borohydride. The compound is a competitive inhibitor with respect to DNA but not ATP. Moreover, the presence of DNA substrate protects the enzyme against this inactivation but the presence of ATP shows no effect. The reduced enzyme . pyridoxal 5'-phosphate complex displays a new absorption maximum at 325 nm and a fluorescence emission at 390-400 nm when excited at 325 nm which are characteristic for epsilon-N-(phosphopyridoxyl)lysine. Thus, B. laterosporus DNase appears to have an essential lysine residue at the DNA binding site of the enzyme, and the enzyme possess two different active sites, a DNA binding site and an ATP binding site.

An Adenosine Triphosphate-Dependent Deoxyribonuclease from &lt;italic&gt;Bacillus laterosporus&lt;/italic&gt;. Improved Purification, Subumt Structure and Substrate Specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.

Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis.

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.

ATP-dependent Deoxyribonuclease Activity in Segregated Cell Types of Caulobacter crescentus

Partially purified bacterial extracts were prepared from segregated stalked and swarmer bacteria of Caulobacter crescentus and assayed for ATP-dependent deoxyribonuclease activity. This activity was found to be very low in the swarmer bacteria compared with a pure population of stalked cells or an asynchronous population of C. crescentus.

A mutant of Pseudomonas aeruginosa deficient in an ATP-dependent deoxyribonuclease.

A mutant of Pseudomonas aeruginosa PAO1 originally isolated on the basis of its sensitivity to methyl methanesulphonate was found to be (i) sensitive to u.v.- and gamma-irradiation, (ii)deficient in recombination as assayed by transduction and conjugation and (iii) deficient in an ATP-dependent deoxyribonuclease activity. Its marker (mms-13) is cotransducible with argB and pyrE which are mapped at approximately 22 min on the P. aeruginosa chromosome.

Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damages in mycobacterium smegmatis.

Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methanesulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methanesulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis. In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism. The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.