Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of gamma-endonuclease from Micrococcus luteus.

gamma-Endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkalistable lesions in gamma-irradiated (N2, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. gamma-Endonuclease Y induces breaks in OsO4-treated poly(dA-dT) and apparently is specific towards gamma-ray-induced base lesions of the t' type. The complete excision repair of gamma-endonuclease Y substrate sites has been performed in vitro by gamma-endonuclease Y, DNA polymerase and ligase.     

Properties of an endonuclease activity in Micrococcus luteus acting on gamma-irradiated DNA and on apurinic DNA

A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against gamma-irradiated DNA was stimulated five-fold with 5 mM Mg++, whereas that against apurinic sites was less dependent on the Mg++ concentration. 100 mM KCl inhibited the gamma-ray endonuclease, but not the apurinic endonuclease activity. In gamma-irradiated RNA the protein recognized 1.65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a Km-value of 73 nM.     

Micrococcus luteus endonucleases for apurinic/apyrimidinic sites in deoxyribonucleic acid. 1. Purification and general properties

Two chromatographically distinct endonucleases from Micrococcus luteus, specific for apurinic and apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from DNA glycosylase, unspecific endonuclease, and phosphatase activities. The two enzymes behave as monomeric proteins of approximately 35000 daltons. In addition to their different chromatographic properties on CM-cellulose, P-cellulose, hydroxylapatite, and DNA--Sepharose, both AP-endonucleases can be distinguished as follows: AP-endonuclease A has an isoelectric point of 4.8, shows a half-life of 4 min at 45 degrees C, reacts optimally at pH 7.5 and has a KM value of 2.3 X 10(-6) M. AP-endonuclease B has a pI of 8.8, is more stable at 45 degrees C (half-life of 10 min), and reacts optimally between pH 6.5 and pH 8.5; its KM value is 3.7 X 10(-6) M.     

Template properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I: indication for a role of apyrimidinic-sites in UV-induced mutagenesis

Ultraviolet irradiation alters the template properties of poly(dC) when replicated by Escherichia coli DNA polymerase I. These effects are due to base modifications. Some of them are identified as apurinic/apyrimidinic sites (AP-sites) by their sensitivity to AP-endonuclease B purified from Micrococcus luteus, and their template properties. The rate of formation of AP-sites in poly(dC) is estimated at 3 X 10(-7) site per nucleotide per J.m-2. Exposure of supercoiled or relaxed pBR322 DNA to UV light results also in the formation of sites sensitive to AP-endonuclease B. In this case, the rate of formation of AP-sites is the same in relaxed or supercoiled DNA: 0.3 X 10(-7) site per nucleotide per J.m-2. The apyrimidinic sites are generated through the processing of an ultraviolet induced primary lesion. We suggest that this lesion is cytosine hydrate by its rate of decay and preferential formation in single stranded DNA. Our results suggest that AP-sites might be a minor pathway leading to UV-induced mutagenesis.     

Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Micrococcus luteus endonucleases for apurinic/apyrimidinic sites in deoxyribonucleic acid. 2. Further studies on the substrate specificity and mechanism of action

Two endonucleases specific for DNA-containing apurinic or apyrimidinic sites (AP-endonucleases A and B) have been isolated from Micrococcus luteus and highly purified. These enzymes have no exonuclease activity. Both AP-endonucleases hydrolyze DNA-containing apurinic or apyrimidinic sites at the 5' end of the lesion, thus generating 3'-hydroxyl and 5'-phosphoryl end groups. DNA-containing pyrimidine dimers, introduced at low doses of UV, are not hydrolyzed, whereas DNA-containing lesions, introduced at high doses of UV or by gamma irradiation are nicked by either AP-endonuclease. During hydrolysis of apurinic DNA, neither of the AP-endonucleases acts as a processive enzyme.     

Occurrence and loss of alkali-labile sites in newly synthesized DNA of UV-irradiated Escherichia coli K12

In pulse-labelled DNA of ultraviolet-irradiated E. coli, alkali-labile sites were detected. They do not occur in undamaged cells. These sites are produced in wild-type cells as well as in uvrA, uvrB and recA derivatives. Restoration of the synthesis of DNA molecules free of alkali-labile sites requires recA products and involves also uvrA and uvrB products. The chemical nature of alkali-labile sites and their biological function are obscure. They might be stretches of RNA that traverse the lesions, blocking DNA replication and priming recA-dependent DNA replication.     

Enzymes from Micrococcus luteus involved in the initial steps of excision repair of spontaneous DNA lesions: uracil-DNA-glycosidase and apurinic-endonucleases.

Uracil-DNA-glycosidase that releases free uracil from single-stranded or double-stranded deaminated DNA and poly d(A-U) has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 16,000 and can be separated from uracil-endonuclease and endonucleases (AP-endonucleases) specific for apurinic and apyrimidinic sites. Uracil-DNA-glycosidase does not act on guanine residues opposite uracil in double-stranded DNA and on xanthine in deaminated DNA. The glycosidase generates apyrimidinic sites which can serve as substrate sites for different AP-endonucleases from M. luteus.     

Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain

The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.     

Enzymatic digestion of DNA gamma-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.     

NAD+ kinase: molecular weight determination by low-angle laser-light scattering

Low-angle laser-light scattering (LALLS) was employed to measure the absolute molecular weight of chicken liver NAD+ kinase (NADK). The weight-average molecular weight (Mw) was found to be 275 000 +/- 15 000. The corresponding value for the second virial coefficient was -1.65 X 10(-3) ml X mol X g2. The value for Mw is in close accord with estimates reported for pigeon liver (270 000) and C. utilis (260 000) NADK. If the active enzyme is a dimer, the weight difference between pigeon/chicken liver and rabbit liver (136 000) NADK would indicate that the latter enzyme is an active monomer unit.     

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.     

Chemistry of the alkali-labile lesion formed from iron(II) bleomycin and d(CGCTTTAAAGCG)

Two sets of products are formed from DNA upon treatment with Fe(II).bleomycin + O2. One set, which is believed to derive from a C-4' hydroperoxy derivative of the DNA deoxyribose moiety, includes the four possible base propenals, as well as DNA oligomers having deoxynucleoside 3'-(phosphoro-2"-O-glycolates) at their 3'-termini. The other set of products consists of free bases and alkali-labile lesions, the latter of which had not previously been characterized structurally. By use of the self-complementary dodecanucleotide d(CGCTTTAAAGCG) having a site modified by Fe-bleomycin three nucleotides from the 5'-end, it has been possible to characterize the alkali-labile product as a C-4' hydroxyapurinic acid. When the bleomycin-treated dodecanucleotide was treated with agents that effected decomposition of the alkali-labile lesion, products of the form CpGpx were obtained, and these proved useful for structural characterization of the alkali-labile lesion. Treatment with alkali produced CpGpx, where x was 2,4-dihydroxycyclopentenone. Alternatively, treatment with hydrazine provided a pyridazine derivative, and aqueous alkylamines led to formation of CpGp itself. The structures of all dinucleotides produced from the alkali-labile lesion were verified by direct comparison with authentic synthetic samples.     

DNA strand scission by activated bleomycin group antibiotics

The bleomycins (BLMs) are a structurally related group of antitumor antibiotics used clinically for the treatment of certain malignancies. The mechanism of action of the BLM is believed to involve DNA strand scission, a process that requires O2 and an appropriate metal ion; the therapeutically relevant metal is probably iron or copper. DNA strand scission by activated Fe X BLM involves oxygenation C-4' of deoxyribose and leads to two sets of products. One set results from scission of the C-3'--C-4' bond of deoxyribose, with concomitant cleavage of the DNA chain. The other set of products consists of free bases and an alkali-labile lesion, the latter of which leads to DNA chain cleavage on subsequent treatment with base. The structures of all of these degradation products have now been established by direct comparison with authentic synthetic samples. Also studied was the activation of BLM with (mono)oxygen surrogates such as iodosobenzene. The chemistry of the activated BLM so formed was remarkably similar to that of activated cytochrome P-450 and structurally related metalloporphyrins, which suggests a mechanistic analogy between the two. Remarkably, both Fe X BLM and Cu X BLM were also shown to be activated by NADPH cytochrome P-450 reductase in a transformation that was dependent on metal ion, O2 and NADPH.     

High ratio of alkali-sensitive lesions to total DNA modification induced by benzo(a)pyrene diol epoxide

The ratio of alkali-labile lesions to total DNA adducts for DNA modified by an active metabolite of benzo(a)pyrene was investigated using DNA sequencing methodology. About 40% of the adducts formed result in alkali-labile sites. About 25% of the lesions were alkali-labile at positions of guanine, 10% at adenine, and 5% at cytosine. This study highlights the potential role of adducts other than the N2-substituted guanine in mutagenic and carcinogenic effects of benzo(a)pyrene.     

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069.

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.     

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.     

Central hypertensive actions of angiotensin I, II and III in conscious rats

The effects of intracerebroventricular administrations of three natural angiotensins, angiotensin I (ANG I 3.8 X 10-11-9.4 X10-10 mol/kg body weight), II (9.6 X 10-12-2.4 X 10-10 mol/kg body weight) and III (2.7 X 10-10 2.5 X 10-9 mol/kg body weight) on systemic blood pressure were investigated in conscious rats. Angiotensin II (ANG II), ANG I and angiotensin III (ANG III), increased blood pressure in a dose-related manner. The order of potency of angiotensins was ANG II greater than ANG I greater than ANG III. The intraventricular administration of a converting enzyme inhibitor (SQ 14225, 6.9 X10-8 mol/kg) abolished the central effect of ANG I, while an angiotensin II analogue ([Sar1-Ala8]ANG II, 1.1 X 10-8 mol/kg) administered intraventricularly inhibited the central pressor effects of these three angiotensins. These results suggest that ANG II is a main mediator of the renin-angiotensin system in the central nervous system.     

Sites of gamma radiation-induced DNA strand breaks after alkali treatment

When DNA is gamma-irradiated in aerated aqueous solution, strand breaks are produced during irradiation or the next few hours. Subsequent piperidine treatment gives rise to further DNA strand ruptures at alkali-labile sites. These different types of DNA chain breaks provoked by gamma-irradiation have been studied with oligonucleotides having defined sequences. The breaks selectively developed inside the DNA chain at alkali-labile sites by piperidine treatment appeared at lower doses preferentially at guanine positions and the order G greater than A greater than T greater than or equal to C was observed. The total contribution of the direct DNA chain ruptures, formed during irradiation and the next few hours, and those obtained by piperidine treatment was studied at doses ranging from 10 to 120 Gy. The chain breaks appeared preferentially at thymine positions and the order T greater than G greater than A greater than or equal to C was shown for the higher doses.