Photoreactivation of the thymine dimer containing DNA octamer d(GCGT TGCG)·d(CGCAACGC) by the photoreactivating enzyme from Anacystis nidulans

Journal of Photochemistry

and Photobiology,

B: Biology, 1 (1988)

323 - 323

323

PHOTOREACTIVATION OF THE THYMINE DIMER CONTAINING DNA OCTAMER d(GCG’l%GCG).d(CGCAACGC) BY THE PHOTOREACTIVATING ENZYME FROM ANACYSTIS NIDULANS J. KEMMINK

Department of Physical Chemistry, Groningen (The Netherlands)

University of Groningen, Nijenborgh

16 9747 AG

A. P. M. EKER

Erasmus University Rotterdam, G. A. van der MAREL Gorlaeus Laboratory, (The Netherlands)

P.O. Box 1736, 3000 DR Rotterdam

(The Netherlands)

and J. H. van BOOM

State University, P.O. Box 9502, 2300 RA Leiden

R. KAPTEIN+

Department of Physical Chemistry, Groningen (The Netherlands) (Received

Keywords.

September

2,1987;

University of Groningen, Nijenborgh

accepted

NMR, photoreactivating

October

16 9747 AG

17,1987)

enzyme,

thymine

dimers, DNA octamer.

Summary Irradiation of the double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with UV light causes dimerization of the two central thymine residues. Proton NMR data reveal that this photodimer has the same chemical structure as the photodimer, which is formed upon UV irradiation of the single strand d(GCGTTGCG), a ck-syncyclobutane-type thymine dimer. Irradiation of the purified thymine dimer double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with visible light in the presence of photoreactivating enzyme isolated from Anacystis nidulans leads to an increase in absorbance at 260 nm, which is characteristic for the repair of thymine dimers. The NMR spectrum recorded after the photoreactivating treatment indeed shows that a complete conversion to the starting octamer has occurred.

+Author to whom correspondence should be addressed. Present address: of Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht, lands. loll-1344/88/$3.50

@ Eisevier

Sequoia/Printed

Department The Nether-

in The Netherlands

324

1. Introduction Preservation of the genetic code carried by DNA plays a key role in the development of living organisms. The genetic material itself, however, is very sensitive to UV radiation and particular chemicals. To overcome the deleterious effects of radiation and chemicals the cell has developed repair mechanisms [l - 31. These repair mechanisms are not perfect and permit some degree of mutation, which is essential for evolution. The main lesions in DNA induced by UV radiation are the cyclobutane dimers formed between adjacent pyrimidines [ 41. Of all possible pyrimidine cyclobutane type photodimers the cis-syn-thymine dimer has been identified as the predominant photoproduct in DNA [ 5 - 81. One of the repair modes, called, enzymatic photoreactivation, repairs specifically cyclobutane dimers of the cis-syn type by absorption of visible or near-UV light. The repair reaction can be represented by the following scheme: PRE + DNA(TT) h-! k

[PRE.DNA(TT)]

-%

PRE + DNA(TT)

+1

in which PRE is the photoreactivating enzyme or photolyase (see ref. 9 for the properties of some photoreactivating enzymes). A study of the photorepair of UV-irradiated oligo(dT), (n > 2) with DNA photolyase isolated from Escherichia coli reveals that the enzyme shows the same activity for oligo(dT), (n = 4 - 18), polythymidylate and native DNA [lo]. The affinity was equally high for all oligomers with n > 7. It was suggested that the critical factor for photoreactivation is recognition of the photodimer by the enzyme and not recognition of distortion in the B-DNA-type structure. Recently we performed extensive two-dimensional NMR studies [ 11, 121 on the double-stranded octamer d(GCGTTGCG).d(CGCAACGC). The NMR spectra showed that the two thymines are indeed present as a cis-syn-cyclobutane dimer. In this paper we show that the photodimer obtained by UV irradiation of the single-stranded octamer is identical with that formed in the irradiated double strand. Fu_rthermore,we report a study on the photoreactivation of this welldefined TT octamer with photoreactivating enzyme isolated from Anacystis nidulans. 2. Materials and methods

2.1. Preparation of the octamer The complementary octameric strands d(GCGTTGCG) and d(CGCAACGC) were synthesized according to an improved phosphotriester method [13,14]. The thymine dimer was prepared by irradiation of the single strand d(GCGTTGCG) with UV light [ll]. After purification of the thymine dimer strand with HPLC methods the complementary strand d(CGCAACGC) was titrated to a 1:l ratio. For comparison one sample of the double-stranded octamer was irradiated with UV light during 1 h.

325

2.2. Pho toreac tiva ting enzyme Photoreactivating enzyme was isolated from the cyanobacterium A. nidulans 1402-l SAUG, grown in a mineral medium [ 151 with white fluorescent lamps as the light source. Cells were disintegrated by passing them twice through a sonic oscillator, followed by centrifugation to remove cell debris. Photoreactivating enzyme was purified by consecutive chromatography on porous silica beads, UV-DNA-cellulose, heparin-sepharose, DEAE-cellulose and DEAE-sepharose. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. The purified enzyme showed, besides protein absorption, an absorption band in the visible region (maximum at 438 nm) which coincides with the action spectrum of photoreactivation. 2.3. Pho toreac tiva ting treatment Samples of 120 pg dimerized octamer and 13.7 pg purified A. nidulans photoreactivating enzyme in 3 ml buffer containing 0.1 M NaCl, 10 mM potassium phosphate pH 7.0, 5 mM 2-mercaptoethanol and 1 mg ml-’ bovine serum albumin was placed in a stirred cuvette thermostatted at 30 “C. After 5 min of dark equilibration the samples were illuminated at 435 nm with a photon flux of 38.2 X 1O-9 einsteins s-l in a high intensity apparatus [16]. The spectral bandwidth was 16.4 nm. Illumination was interrupted in order to record absorption spectra of the photoreactivated sample with a Pye-Unicam SP7-500 spectrophotometer. 2.4. NMR spectroscopy After irradiation the PRE-octamer samples were collected and lyophilized. A ‘H NMR spectrum was recorded on a Bruker HX-360 spectrometer operating at 360 MHz. For comparison also NMR spectra of the UVirradiated double-stranded octamer and the purified thymine dimer doublestranded octamer were recorded.

3. Results and discussion Figure l(A) shows part of the 360 MHz ‘H NMR spectrum of the double-stranded octamer, UV irradiated for 1 h. Two characteristic regions of the spectrum are plotted: the base proton region (H6 for pyrimidines and H8 for purines) and the thymine methyl group region. Lines with low intensity at 6.65 and 0.71 ppm show that the sample contained a photoproduct for about 3% and unreacted octamer for the remaining 97%. Figure l(B) shows the same spectral region of the double-stranded octamer containing the thymine dimer, which had been produced by UV irradiation of the single strand. Comparison of the lines at chemical shift positions 6.65 and 0.71 ppm indicates that the same photodimer must be formed by UV irradiation of the single- and double-stranded DNA. Previous two-dimensional NMR studies have shown that this photoproduct is the cis-syn-thymine dimer.

326

85

80

75

10

65

(PPM)

20

15

10

05

Fig. 1. ‘H 360 MHz NMR spectra of the base proton and methyl group region of (A) the UV-irradiated double-stranded octamer (97% unreacted and 3% thymine dimerized octamer), (B) the purified thymine dimer double-stranded octamer and (C) the thymine dimer double-stranded octamer after photoreactivating treatment. The two peaks marked with X belong to impurities.

In order to examine whether this thymine dimer containing oligonucleotide is recognized by photorepair enzymes we have studied the photoreaction of this octamer with the PRE from A. nidulans. This reaction can be monitored by measuring the absorbance of the PRE-octamer mixture at 260 nm, where the purine and pyrimidine bases of DNA normally show an absorption maximum [17]. Pyrimidine dimers, however, do not show significant absorption at this wavelength, because the C5-C6 bond in the pyrimidine ring is saturated. When the thymines are monomerized the 260 nm absorption band reappears and an increase in absorption at 260 nm can be observed. As shown in Fig. 2, illumination of the PRE-octamer mixture with 435 nm light leads to a rapid increase in absorption at 260 nm, indicating that monomerization of the thymine dimer has occurred. Illumination of the octamer without the PRE present did not lead to a significant change in absorbance at 260 nm. In order to obtain more specific information about the resulting structure after photoreactivating treatment the samples were collected and

327

008 006 0.0 4 0.02

10

20 TIME fminl

30

LO

Fig. 2. Monomerization of the thymine dimer present in the double-stranded octamer d( GCGl+I’GCG).d( CGCAACGC) as a function of illumination time ([PRE] = 9 x lo-* M; [DNA] = 8 x lo+’ M).

examined by NMR spectroscopy. In Fig. l(C) part of the NMR spectrum of the thymine dimer duplex treated with 435 nm light and PRE is shown. Comparison of Figs. l(A) and l(C) in combination with the UV data of Fig. 2 shows that the photoreactivating treatment leads to formation of the starting octamer. Therefore, we can conclude that the photoreactivating enzyme from A. nidulans is able to recognize the thymine dimer in the octamer d(GCGTTGCG).d(CGCAACGC) and to monomerize the dimer using visible light yielding the starting octamer d(GCGTTGCG).d(CGCAACGC). Previously, we have shown that the octamer duplex does not undergo major conformational changes upon thymine dimer formation [ll, 121. For instance, all imino protons including those of the thymines are involved in Watson-Crick-type hydrogen bonding [ 121. Therefore, it is likely that the PRE recognizes the thymine dimer itself rather than a disruption of the DNA structure. This is in agreement with conclusions of Jorns et al. [lo] based on their study on thymine dimers in single-stranded (dT), oligomers.

Acknowledgments This work was supported by the Netherlands Foundation Research (SON) with financial support from the Netherlands for Advancement of Pure Research (ZWO).

for Chemical Organization

References 1 P. C. Hanawalt, Repair of genetic material in living cells, Endeavor, 87.

31 (1972)

83 -

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