The action of a mammalian endonuclease on psoralen-bound DNA

The action of a mammalian endonuclease on psoralen-bound DNA

Chem.=Biol.Zntenactions,31(1980) 179-188 0 EIeevier/Nortb=HollanaScIentIfIcPubIisberaLtd. 179 THE ACTION OF A MAMMALIAN ENDONUCLEASE ON P!SORALENBOU...

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Chem.=Biol.Zntenactions,31(1980) 179-188 0 EIeevier/Nortb=HollanaScIentIfIcPubIisberaLtd.



Department of Radiaiion Oncology, Department of Pathology and aSchool of Medicine, UCLA Center for the Health Sciences, Lee Angeles, CA 90024 (U.S.A.) (Received February 28th. 1979) (Revision received February 2Sth, 1980) (Accepted Februery 26tb, 1980)


The sequential actions of two enzymes believed to be involved in DNA repair, namely a mammalian endonuclease ZWJ.the bacterial DNA polymerase I on psoralen bound ‘“P-labeled DNA, was studied. When ultravioletirradiated DNA is exposed to the sequential action of the endonuclease, the formation of single-strand breaks prepares the DNA for the exonucleolytic excision of thymine dimers. The mammalian endonuclease purified from rat liver to electrophoretic homogeneity is inactive on normal DNA, DNA irradiated at 360 nm or DNA mixed with psoralen without irradiation. Incubation of psoralen-bound DNA labeled with “P with the endonuclease releases the isotope in the a&-l soluble indicating that psoralen-bound DNA is susceptible to the endonucleolytic attack. Sedimentation of DNA on sucrose gradients indicates that there is no collapse of the DNA molecule after treatment with the endonuclease, Moreover, there is no release of the adduct in the acid soluble after treatment with DNA polymerase, indicating that the 5-3-min exonucleolytic activity of that enzyme is impaired by the remaining crosslinks. The crosslinks also inhibit the incorporation of i3H] dATP in presence of DNA polymerase I.


Uneven pigmentation is of cosmetic concern to many. In India several centuries ago leukoderma was treated with a poultice of Psuxdea corylifolia [l-3]. Egyptians treated vitilligo with an extract of a weed [4], the Ammi majus of the Umbillifera genus, which grows in the Nile Valley. Although the medicinal preparation of the crude plant was known to medieval Arabs and 19tb century German physicians, the active agents, psoralens and furocoumarins, were only isolated in 1841 and synthesized in 1933 [ 5] . The 8=

180 methyoxyp&:~ralenwas crystallixed in 1972 161. Furocoumarins form cyclobutane monoadducts with the thymine and cytosine bases of the DNA in the presence of ultraviolet light at 365 nm [7,31 The formatimi of crosslinks occurs in three steps. In the first, in absence of light, the ~furocoumarins bind to DNA through weak forces. In a second step, which is activated by ultraviolet light, covalent bonds are formed between the furo~~umarin and the pyrimidine bases. The pyrimidine bases react with their 5,6 double bond and the furocoumarin with either their 3,4 or their 4’ 5’ double bond. Whenever the bonding between the pyrimidine and psoralen involves the 3,4 double bond of the latter, no further absorption of light takes pIace, but if it involves the 4’ 5’ double bond, three additional quanta of radiation energy are absorbed at 365 nm, and a reaction with a pyrimidine base located on the complementary strand involving the 3,4 double bond takes place. These double-strand crosslinks are stable to heat and alkali [ 7-9 1. Yoakum and Cole [lo] have shown that psoralen and light treatment of plasmids ColEl and ColEl-amp relax supercoiled twists in the covalently closed molecules. On the basis of these findings, they postulate a new mechanism for the preferential binding of psomlen to the non-nucleosomal DNA of mammalian chromatin. They propose that the positive super-coiling of the nucleos~& DNA and the presence of histones contribute to interference with biiding. Work from several laboratories, including those of Reid and Walker [ll] , Yin et al. [12], Chat&a et al. [ 133, Ben-Hur and Elkind 114,151, have shown that psoralen-bound DNA is rapidly repaired in bacterial and mammalian ills. In 1373 Cole [ 163 pro~sed that in normal cells the repair of crosslinks formed in psoralen-bound DNA depends won excision repair and recombination function. In a first step, an excision repair endonuclease makes two cuts in one strand of the DNA close to one site of binding of the diadduct. This process yields a monoadduct on one strand and a gap on the other. The monoadduct blocks the action of DNA polymerase and thereby prevents patching of the strand containing it. To repair the monoadduct, the excision gap on the opposite strand is widened by a nuclease and the extended gap is filIed by strand exchange with an homologous duplex; thus filling the gap with a sequence comp1em.enter-y to that containing the monoaddict. Excision rep& of the monoadduct completes the repair sequence. On the basis of experiments in which tritiated angelicin and tritiated psoralen were used, Bordlin et al. [17] have suggested that ihe excision mechanism of the monoadduct is different from that of the diaLduct. Thus, existing models postulate that the first step in repair involves a repair endonuclease, and that because of the presence of the diadduct the exonucleolytic attack and the polymerizing action of DNA polymerase I is inhibited after nicking witb the endoriuclease. Excision repair endonucleases have been purified from bac&rial and mammalian cells (for review Refs. 18 and 19). l

181 An endonuclease acting on ultraviolet-irradiated DNA to which ultimate carcinogensof AAF and ‘I-bromoanthracene have been bound has been purified to electrophoretic homogeneity [23]. The enzyme is partMy magnesium dependent and its activity is lost by freezing. It has no activity on non-injured, single-stranded, or a.purinic DNA. After homogenization of liver t,issue followed by tissue fractionation, only negligible levels of enzyme activity are found in cellular organelles. Thus, all crude enzyme activity is found in the high-speed supernatant in rat liver. Two findings suggest that the enzyme plays a significant role in repair of damaged DNA; it is induced by the injection of carcinogens (Van Lancker and Tomura, unpublished), and when antiserum prepared against rat liver enzyme is added to rat liver cytosol, more than 80% of the excision repair endonucleolytic activity is abolished (Van Lancker and Tomura, unpublished). Inasmuch that models for repair of crosslinks postulate a first step catalyzed by an excision repair endonuclease, the ability of the purified endonuclease to act on psoralen-bound DNA was investigated. Since at least in the case of mono., adducts, the endonucleolytic step can be followed in vitro by the exonucleolytic action and patching action of DNA polymerase I [23 1, the effects of the polymerase on psoralen-bound DNA were also investigated. METHODS

For the purpose of labeling the DNA, partially hepatectomized rats [ 201 were injected [23] with I-I3 32P04 at 18, 20 and 22 h and sacrificed 24 h after partial hepatectomy. After purification by the Marmur method [ 21,241) a solution of 32P-labeled .QNA (20 pgglml) was allowed to mix with psoralen (0.1 pg/ml) for 2 h in the dark. The mixture was then exposed for 3 h to near ultraviolet light (360 nm) generated by two tubular fluorescent sunlamps (Burton Division Cabaton Corp. 9100). The DNA solution was spread in a Petri dish. The thickness of the film did not exceed 2-3 ‘mm. The distance between light source and target was 30 cm. The temperature denaturation curve suggests that extensive crosslinking takes place under those conditions (Fig. 1). The mammalian endonuclease used in these experiments was purified to electrophoretic homogeneity by methods described in detail [ 231. DNA polymerase I was purified from E. cdi and determined by the method of Lee-Huang and Cavalieri [22] . The alkaline phosphatase was purchased from Worthington Biochemical Corporation. The number of single-strand breaks introduced hi the endonucltsase was measured by determining the amount of 32P released from DNA after incubation of the psoralen-bound DNA with endonuclease and alkaline phosphatase. Sedimentation of DNA on neutral and alkaline sucrose gradients has been described in detail [ 231. RESULTS When



enzyme expressed


of IzP released is plotted on the ordinate versus the pg protein, it is clear that after the sequential attack by

0.20 1

I -&


I 90


Fig. 1. Native DNA was treated with psoralen and exposed to near ultraviolet light as described in the Metilode section. DNA was diluted with 0.15 M saline in get approx. 0.2 0-D. units in a l-cm high uath at 260 nm. Then the hvuerchromicitv on the DNA -_ was determined contir~uousl~ from 40°C to 100°C at 260 nm with a Gilford recorder 1331.

endonuclease and alkaline phosphatase, the 32P released increases with the concentration of enzyme in the incubation mixture. Thus, psoralen DNA is a substrate for the endonuclease and the incidence of strand breaks increases with the amount of enzyme used. When a substrate containing thymine dimers of acetylaminofluorene isequentially treated with our purified endonucle&qe, alkaline phosphatase and DNA polymerase, the exonucleolytic activity of the polymerase releases acid soluble fragments which contain either thymine dimers or acetylaminofluorene adducts [23]. In contrast, when psoraleu-bound DNA is subjected to the sequential action of these enzymes, no acid soluble component interferes with the exonucleolytic activity of the polymerase I and prevents the release of the monoadducts as well as that of the diadducts. In Pig. 2 we show the activity of DNA polymerase I using calf thymus DNA as a substrate. The incorporation of r3H] dATP in DNA is almost





polymerose in units

Fig. 2. Priming ability of the near-ultraviolet exposed DNA for DNA polymerase. DNA was exposed to 360 mp in presence or absence of psoralen as described in Methods, then treated by ultravioletendonuclease and alkaline phosphatase as previously described [ 23). The DNA was purified as described and used as a substrate for DNA polymerase I. The incorporation of [‘H]ATP into the acid-soluble fraction {ordinate) L expressed as a function of the units of enzyme used (abscissa).

tottiy inhibited when psoralen-bound DNA is used as a substrate. A finding confirming the prediction of Cole [ 161. Ou et al. [25] also used psoralen DNA as a substrate for DNA polymerase I, but without previous treatment with endonuclease, the enzyme activity was inhibited 9’7%. Duplex DNA untreated with endonuclease is a weak template for DNA polymerase I. The incorporation of labeled triphosphate is less than 10% of the incorporation in ultraviolet-irradiated DNA, The incorporation of in duplex DNA treated with psoralen and light but not nicked with the endonuclease are undetectable. A finding in agreement with that of Ou et al. 1251. The sedimentation properties of the crosslinked DNA treated with the endonuclease in neutral and alkaline sucrose were also investigated. DNA prepared from rat liver was divided into two batches: one unexposed to psoralen (control), another exposed to psoralen. The latter was ultraviolet-

164 cpm




AND 360 X

-b-treated o---e Treated

10 Fraction



Fig. 3. Sedimentation

on neutral or alkaline sucrose gradient. DNA (5 rg) in all cases placed on a neutral or alkaline sucrose gradient (5-20%) and centrifuged after the method of Buxgi and Hershey [21,34 ]. Fractions were collected and their radioactivity measured as described in Methods. Psoralen-bound DNA and calf thymua DNA were either untreated or treated with the purified endonuclease prior to sedimentation. (e ---e)

irmdiated at 360 nm. !Samples of both types of DNA were placed on top of neutral or alkaline sucrose gradients without treatment or after treatment with the purified mammal& endonuclease. When ultraviolet-irradiated DNA or ar%ylaminofluorene-bound DNA are treated with the e:ndonuclease and placed on alkaline sucrose gradients, the peak of sedimentation is displaced toward the lower concentration of sucrose [23]. No such displacement takes place when psoralen-bound DNA is treated with the endonuclease (Fig. 3) probably because of extensive crosslinking.


CONCLUSION Although there is evidence that crosslinks can be repaired in mammalian cells, the molecular mechanism of such repair is unknown. The most plausible is that proposed by Cole [ 16 ] ; it implies that the psoralen-bound DNA can be attacked by a repair endonuclease, but in contrast to what happend when thymine dixners are formed, psoralen-bound DNA cannot serve as a substrate for DNA polymerase I. We have previously shown that thymine dimers and acetylaminofluorene monoadducts are removed from DNA by the sequential action of the mammalian endonuclease used here, and DNA polymerase I. The purpose of this study was to investigate the susceptibility of psoralen-bound DNA to be nicked by the endonuclease. The study in which [JZP]psoralen-bound DNA was used as a substrate clearly shows that the endonuclease causes singlestrand breaks in the psoralen-bound DNA in vitro. Whether it operates as the first enzyme in the sequence of steps involved in crosslink repair in viva is not known, but cannot be excluded. Bowever, in keeping with Cole’s hypothesis, the nicked DNA inhibits DNA polymerase I, and therefore even the monoadducts cannot be repaired in vitro by the enzymic sequence used to repair thymine dimers or acetylainofluorene adducts. Several investigators have measured the number of either monoadducts or crosslinks or both using either electron microscopy [26-291 or [3H] psoralen [ 25,30-321. Because of the unavailability of radioactive psoralen it was impossible to determine how many monoadducts or crosslinks were formed in our experiments. Such measurements would have been of interest and are planned for the future. However, because the specific activity of the ]j*P]DNA used as substrate and the amount of 32P released after incubation with the endonuclease is known, it is simple to calculate the number of single-strand breaks formed: 3.25/1000 nucleotides when 5 pg of purified enzyme are used in the incubation mixture. Although an exact ratio of single-strand breaks to number of adducts cannot be calculated, some conclusion as to the restriction to the action of the endonuclease can be reached. Let’s consider data obtained on calf thymus under experimental conditions similar to ours. Dall’Acqua et al. [31] report 7.4 adducts formed per 1000 nucleotides. Ben Hur and Elkind [14] found that 11% of the psoralen DNA adducts are in the form of crosslinks. For the sake of simplicity let us assume that 10 adducts are formed per 1000 nucleotides, 10% of which are crosslinks. A mechanism proposing that monoadducts are repaired by a single endonucleolytic break followed by excision and patching would require at least 9 single-strand breaks per 1000 nucleotides. If in addition one crosslink is present and the mechanism proposed by Bordln et al. [17] for crosslinks repair obtains, then 3 additional single-strand breaks per 1000 nucleotides are expected. Thus, a total of 12 breaks per 1000 nucleotides. Present data shows that the incidence of single-strand breaks is too low to






in pg of protein

P’ig. 4. Phosphorus release in WMole (ordinate) as function of enzyme concentration (;kscissa) in ccg of protein. DNA ultraviolet-irradiated in presence of psoralen (e---e) and DNA mixed with psoralen (o----o ) without ultraviolet-irradiation. The substrates were prepared as described in the Methods section. The purified endonuclease contained 5 U of activity per 0.25 rg of protein. Under the conditions of the experiment no relezue of 3zP w& detected when labeled DNA was irradiked at 360 nm and treated with tha endonuclease.

account for repair of dl monoadducts and crosslinks. This contrasts with results obtained with acetylaminofluorene-bound DNA [ 231. When acetylaminofluorene-bound DNA is used as a substrate with the purified endonuclease, the number of strand breaks correlates closely with the number of adducts formed. Moreover, 99% of the adducts are removed when the endonuclease attack is followed by that of alkaline phosphatase and DNA polymerase [ 231. As shown in Fig. 4, the endonuclease attacks DNA irradiated in presence of psoralexl. The site of the endonucleolytic cut cannot be determined from this experiment. Because of the action of the enzyme on monoadducts like acetylaminofluorene, it seems, however, fair to assume that part of the endonucleolytic activity is directed toward the monoadducts. If this is thie ctie and since exonucleolytic excision by DNA polymerase is inhibited, it would appear that crosslinks may inhibit. the endonucleolytic step of repair. More direct evidence for this mode of action will be sought by the use of tritiated psoralen and DNA of known sequence.


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