The formation, identification, and significance of DNA ... - Europe PMC

Br. J. Cancer (1987),

55, Suppl. VIII, 135-140

Br. J. Cancer (1987), 55, Suppl. VIII,

135-140

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The formation, identification, and significance of DNA-protein cross-links in mammalian cells Nancy L. Oleinick, Song-mao Chiu, Narayani Ramakrishnan & Liang-yan Xue Division of Biochemical Oncology, Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. Summary DNA-protein cross-links (DPC) are formed by a variety of radiations and chemicals which act via free radical formation. Covalency is inferred from the resistance of the cross-links to harsh treatments. In mammalian cells, a background of DPC (6000 per V79 cell) may result from normal associations of chromosomal loops with the nuclear protein matrix. After ionizing radiation, the elevated level of DPC (150 per Gy per V79 cell) are enriched in actively transcribing DNA and in a subset of proteins of the nuclear matrix. DPC formation is reduced by hydroxyl radical scavengers, by oxygen, and by hypertonic medium and is enhanced by hypotonic medium and by removal of intracellular glutathione. DPC are repaired more slowly than single-strand breaks and not at all when formed during metaphase. During the postirradiation period, changes in the sequence composition of the DNA of residual DPC are consistent with the preferential repair of DPC in actively expressed genes. Excision repair mechanisms have been proposed. Unrepaired DPC may block normal functions of the nuclear matrix, such as replication and transcription.

effected. Extraction with organic solvents, equilibrium density gradient centrifugation in CsCl, sedimentation of protein with DNA, gel filtration through large pore molecular sieves, and the binding of protein but not free DNA to glass fiber or nitrocellulose filters have all been employed. Although less sensitive than alkaline elution, separation techniques provide a better estimate of the amount of DNA cross-linked to protein and the possibility of isolation of the complexes for characterization. Not all techniques will be applicable to all cross-linking agents. For example, Strniste and Rall (1975) were able to quantify the protein bound to DNA after UV irradiation of chromatin by chromatography on Sepharose. We have found this method to be less reliable for measurement of DPC after y-radiation, because the coincident strand breaks result in some fragments of DNA which are small enough to elute with protein. In our laboratory, a filter-binding technique has been the most useful (Chiu et al., 1984, 1986a). When whole cells, nuclei, or pre-isolated complexes are solubilized in 1 M NaC104, 1% Sarkosyl and passed through a nitrocellulose filter, a large percentage of the protein becomes bound to the filter. Free DNA, either single- or double-stranded, does not bind; the only DNA which is retained by the filter is that which is tightly associated with protein. This method is simple, allows for quantification of the number of DPC present, and permits recovery of the DPC from the filter for biochemical analysis. Accurate measurements of DPC, however, generally require a minimum radiation dose of 5-10 Gy. A major reason for the inability to quantify DPC at lower doses is the presence of a background level of DPC in untreated cells (usually 1-3% of the DNA bound to protein). With alkaline elution, this background is not readily apparent, because the DNA of untreated cells is retained on the filters by virtue of its large size irrespective of the presence of cross-links. The background DPC are a natural consequence of normal metabolism, and both normal and damage-induced DPC need to be characterized to fully understand the significance of each type in cells. We have asked the following three questions: How many DPC are there? What proteins are involved? And what DNA sequences or classes are bound to protein?

DNA-protein cross-links (DPC) are generally defined in an empirical fashion. When cells or sub-cellular constituents, such as chromatin, are treated with a wide variety of DNAdamaging agents, a fraction of the DNA and protein resists the normal procedures for separating them. The tight association of DNA and protein is maintained in spite of harsh treatments, such as alkaline solutions, detergents, organic solvents, and strong denaturing agents (Cress & Bowden, 1983), lending evidence in support of covalent bond(s) linking DNA and protein, although the chemistry has been defined in only a few model systems. Thus, a thymine-cysteine adduct was isolated from UV-irradiated bacteria by Smith (1970), and other adducts have been identified after irradiation of solutions of nucleotides and proteins or amino acids. The only case in which the chemistry of a covalent linkage between mammalian genomic DNA and protein has been defined is the phospho-tyrosine linkage between topoisomerase II and its cleaved DNA substrate; this structure, an intermediate in the strand passing reaction of topoisomerase II, accumulates when the enzyme is inhibited by intercalating agents, such as m-AMSA (Nelson et al., 1984). DPC are formed as a result of treatment of cells with ionizing or ultraviolet radiation, visible light in the presence of a photosensitizer and oxygen, chemical oxidizing agents, metals and metal complexes, DNA alkylating agents, intercalators, and agents which deplete intracellular thiol levels. Since the definition of DPC is empirical, study of these lesions depends upon the methods for their measurement. A common method of demonstrating the presence of DPC in cellular DNA is alkaline elution (Fornace & Little, 1977). Cells are lysed on a filter, and an alkaline solution is percolated through the lysate. Broken DNA single strands are eluted, while the majority of intact chromosome-size DNA remains on the filter. The elution of fragmented DNA is impeded by the presence of DPC and accelerated by proteinase K. The major advantage of alkaline elution is that detection of DPC is possible at doses of radiations or chemicals below those needed by most other means. However, it permits neither precise quantitation of the number of DPC nor isolation of DPC for biochemical analysis. Other techniques for the measurement of DPC depend upon the physical separation of DNA from protein due to differences in size, density, solubility, or binding properties. When cross-links are present, clean separation is not

Biochemical characterization of DNA-protein cross-links

Correspondence: N.L. Oleinick.

To answer the first question, DPC of Chinese hamster V79

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N.L. OLEINICK et al.

cells were recovered from the nitrocellulose filters and digested with proteinase K, and the size of the DNA was determined on agarose gels under non-denaturing conditions (Ramakrishnan et al., 1987). Based on the size of the fragments (12.2 x 106 Da), the amount of DNA in DPC, and the size of the haploid Chinese hamster genome (3.0 x 109 bp, each cell containing 5-6 pg of DNA), we calculated that each untreated cell in exponential growth contains approximately 6000 DPC. When cells were given up to 100 Gy of yradiation, the size of the cross-linked fragment remained the same but the number of DPC increased linearly by 150 per Gy. Thus, an accurate measure of damage-induced DPC requires a dose of damaging agent that will give a significant number of DPC above the background of 6000. We next asked whether at higher radiation doses, it would be possible to involve all of the nuclear DNA in DPC or whether a saturation value would be reached. We observed that above 2-300Gy fewer DPC were obtained per Gy, the average size of the recovered fragments decreased, and the maximum number of DPC measured was 6-7 x 104. These results indicate that all nuclear DNA may not be equally susceptible to DPC formation. They also suggest that the DPC may be related to the anchorage of chromosomal loops to the nuclear protein matrix, since it has been estimated that there are approximately 6-12 x 104 such anchorage sites per cell (Mullenders et al., 1983). The second question concerned the proteins cross-linked to DNA. Studies with various model systems have shown that any molecule can become crosslinked to DNA if free radicals are generated in the solution (Smith, 1976). This includes proteins normally associated with DNA, such as histones of the nucleosomal core (Mee & Adelstein, 1981), and proteins a

b

which do not contact DNA in living cells, such as albumin or lysozyme (Minsky & Braun, 1977). The proteins involved in DPC of intact cells have been analyzed by isolation and gel electrophoresis of various protein fractions, and, in some cases, by immunoassay of Western blots. Olinski et al. (1981), Banjar et al. (1983) and Wedrychowski et al. (1985) investigated the proteins cross-linked to DNA by chromium salts, BCNU, cis-platinum, and y-irradiation. A complex mixture of chromosomal and nuclear matrix proteins was observed by reaction of Western blots of the separated proteins with antibodies raised against various nucjear protein fractions. Both our laboratory (Oleinick et al., 1986; Chiu et al., 1986) and that of Cress (1985) have identified a subset of nuclear matrix proteins as the dominant species cross-linked to DNA in normal and y-irradiated mammalian cells (Figure 1). The major silver-stained band of the cross-linked proteins is found at 37 kDa. Close inspection reveals the presence of a pair of bands at 37 and 41 kDa. In various preparations, the relative amount of staining material at 37 and 41 kDa has varied. Other proteins, both larger and smaller, are present. Most of these are also observed in lanes of the gel containing total nuclear matrix proteins and in lanes containing the proteins recovered from the DNA peak of a CsCl gradient. The band near 170 kDa is probably topoisomerase (Miller et al., 1981). When DNase I is not used in the preparation of the proteins for electrophoresis, the topoisomerase band is not as prominent as in the preparations shown here. The bands near 40 kDa are always observed, whether or not DNase I has been included. This suggests that not all the linkages between DNA and protein are covalent or that only a short oligonucleotide remains

c

d

e

f

kDa

g -

_ 170

200.0

- 116.0 -92.5 - 66.2

BSA

-66 59

55 -

45.0

-

31.0

-441

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2

26 - 21.5

Figure 1 Gel electrophoretic analysis of nuclear matrix proteins and of proteins cross-linked to DNA of Chinese hamster V79 cells. (b) Cultures were given 100Gy, and dehistonized nuclei were digested with EcoRI. The nuclear matrix, with about 5% of nuclear DNA, was collected by centrifugation. (c, d) Cross-linked proteins

were

prepared by the lysis of cells in

1 M NaClO4,

1%

Sarkosyl. The DNA-protein complexes (>98% of the DNA and 6-7% of cellular protein) were pelleted at 100,000g, resuspended in the same solution, and filtered through nitrocellulose. The filter-bound material (40-50% of input protein and 2% and 10% of input DNA from 0 (c) and 10OGy (d), respectively) was recovered. (e, OGy; f, 1OOGy) The DNA-protein complexes were banded in CsCl, and the DNA peak was collected, dialyzed, and concentrated. After digestion of samples c-f with DNase I, the proteins were dissolved and subjected to electrophoresis in 7.5% polyacrylamide gels containing SDS and observed by silver staining. The densely staining band of M, 66.2 kDa in c-f is bovine serum albumin (BSA) which was included in the DNase I digestion buffer to stabilize the enzyme. (a, g) Molecular weight markers.

CHARACTERIZATION OF DNA-PROTEIN CROSS-LINKS which does not significantly alter the mobility of the protein. Importantly, some histones may be present, but they are not the dominant species for DPC of growing cells. We have addressed the question concerning the sequences of DNA found in DPC of control and treated cells by recovering the DPC, removing the protein with proteinase K and chloroform-phenol extraction, then hybridizing the denatured DNA to probes of transcriptionally active or inactive sequences. The DNA derived from DPC of untreated V79 cells was highly enriched in sequences hybridizing to poly(A+)RNA and to ribosomal RNA (Chiu et

al., 1986a). Furthermore, the additional DNA cross-linked

to protein by y-radiation, but not by UV-radiation, was also enriched in actively transcribing sequences. We have recently explored the relative susceptibility for DPC formation in mouse

,B-globin

sequences as a

function of their transcrip-

tional activity and sub-nuclear location (Ramakrishnan et al., in preparation). The globin sequences were investigated in (a) L-929 fibroblasts, in which the globin genes are insensitive to micrococcal nuclease and DNase I, not matrixassociated, and not transcribed; (b) mouse erythroleukaemia (MEL) cells, in which globin genes are DNase I-sensitive, moderately m. nuclease-sensitive, but not matrix-associated and not transcribed; and (c) MEL cells induced to synthesize globin with hexamethylene-bisacetamide (HMBA). In the latter, globin genes are sensitive to both DNase I and m.

nuclease, matrix-associated, and transcribed. Only in this last state are the globin genes hypersensitive to DPC formation by ionizing radiation. Thus, DPC appear to be formed prefentially in regions of the genome in which chromatin structure is unwound, the genes are near a site of attachment to the nuclear matrix, and the sequences are actively being transcribed.

The mechanism of formation of DNA-protein cross-links Mechanistic studies on DPC formation have been conducted for radiations and chemicals. The yield of DPC is reduced when ionizing radiation is delivered in the presence of scavengers of hydroxyl radicals (Mee and Adelstein, 1979; Chiu et al., 1986b). Thus, OH appears to be a primary initiating species both in cell-free model systems and in living cells, leading to the formation of a free radical on DNA or protein. The yield of DPC is greater for irradiation under hypoxic conditions (Fornace & Little, 1977; Meyn & Jenkins, 1984; Mee & Adelstein, 1985). This suggests that the DNA radical reacts faster with oxygen than with an adjoining protein. To fully evaluate the significance of the difference between the yields of DPC produced in hypoxic and euoxic conditions, it will be necessary to know whether the biochemical constitution of the two types of DPC is the same or different. One possibility is that a change in chromatin structure in hypoxic cells permits a new class of DNA sequences to become available for DPC formation. Alterations of chromatin structure through changes in the tonicity of the medium have been shown to modify the yield of DPC produced by y-irradiation in air (Chiu et al., 1986b). When V79 monolayers were exposed to hypertonic medium, causing the chromatin to condense, the yield of DPC was reduced below that for irradiation in isotonic medium. Since the ability of the DNA in the remaining DPC to hybridize to poly(A + )RNA was also reduced, we concluded that the effect of hypertonic medium was greatest in previously

opened regions of chromatin containing transcriptionally active DNA sequences. In hypotonic medium, which allows an opening of condensed chromatin, the yield of DPC was enhanced, and the enrichment of the cross-linked DNA for active sequences was greater than with DPC formed in isotonic medium. However, there was no difference in either the yield or the active gene enrichment of DPC formed by UV-irradiation of cells treated with hyper-, iso- or hypotonic medium. These results emphasize the need for understanding

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the variations in mechanism of damage induction and in intranuclear environment which can alter the yield of DPC and other DNA lesions. Another important factor in the formation of DPC is the level of intra-cellular thiols. Grunicke et al. (1973) demonstrated that thiol-reactive agents, such as N-ethylmaleimide, iodoacetamide, and arsenite, elevate the level of DPC in Ehrlich ascites cells. Removal of protective thiols may permit metabolically generated radicals to produce a higher level of DPC or some of the adducted proteins may be better substrates for the formation of DPC. Biochemical analysis of the DPCs should help distinguish between these mechanisms. Some evidence for the protective effects of glutathione (GSH) has been obtained (Biaglow et al., 1986). When human lung carcinoma cells (line A-549) were treated with 0.1mM L-buthionine sulfoximine (BSO) for 3-4 days, the GSH level was reduced to non-detectable levels. Under these conditions, the background level of DPC doubled, the radiation dose response for the formation of DPC was unchanged, and the rate of repair of the radiation-induced DPC was markedly reduced. Further study of the role of GSH and other intracellular thiols in DPC formation and repair is needed. A scheme which incorporates some of the considerations mentioned above, as well as others, is presented as Figure 2. A low background level of DPC in normal untreated cells may result from the anchorage of chromosomal loops to the nuclear matrix at sites near regions undergoing transcription or replication. The level of DPC is elevated by a wide variety of radiations and chemicals, many of which lead to the production of excess hydroxyl or other radicals (e.g., radical formation by N-acetoxy-N-acetyl-2-aminofluorene; Rayshell et al., 1983). Peak et al. (1985) showed that the action spectrum for DPC formation by visible light was similar to the absorption spectrum of porphyrins, and the formation of DPC was absolutely dependent upon the presence of oxygen. The mechanism of this effect likely involves the absorption of light by cellular porphyrins and the formation of active singlet oxygen, which may react with DNA or protein directly or through the intermediate formation of other (water) radicals. Alkylating and other DNA-damaging chemicals also may interact directly with DNA or indirectly via water radical formation. Figure 2 also displays the concept that the mechanism of formation of DPC by UV-irradiation differs from mechanisms involving the diffusion of radicals or other reactive chemicals to cellular chromatin. UV-radiation is absorbed directly by DNA bases, and the distribution of UV-induced lesions in the DNA appears to be unaffected by chromatin structure (Williams & Friedberg, 1979; Niggli & Cerutti, 1982; Chiu et al., 1986b). UV-induced DPC are not enriched in actively transcribing DNA sequences (Chiu et al., 1986a). Thus, the characteristics of y-radiation-induced DPC which we have observed are not a result of the techniques for determination of DPC but a property of ionizing radiation (and other radical-generating agents). The evidence argues that nuclear matrix-associated DNA sequences are more susceptible to chemical attack, because those DNA regions are relaxed from higher order chromosomal coiling and because they are located within a more highly hydrated region of the nucleus. Domains of chromatin which are accessible to DNA and RNA polymerases, nucleoside triphosphates, and transcription modifiers may also be most accessible to other soluble molecules (OH', alkylating agents) which must diffuse to the DNA target. If these concepts are correct, we would predict that free radical damage other than DPC would also form preferentially in active, matrixassociated DNA. In fact, we have shown that y-radiationinduced single-strand breaks (SSB) are more abundant in active DNA than in the bulk DNA of mouse (Chiu et al., 1982) and Chinese hamster (Oleinick et al., 1984) cells, and evidence exists for the preferential binding of carcinogenic chemicals to the nuclear matrix (e.g., Hemminki & Vainio, 1979).

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N.L. OLEINICK et al.

~ ~ ~ ~X-ray 0l2

Visible light +

Oxidizing

DNA Alkylating

L

agents

X

agents

0

H

02

DNA-OO

H20

DNAL

DMSO

GSH

Protein

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Base damage Strand breaks

NEM IAA

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Transcription

photosensitizer

+ 02

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-

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BSO

y-Glu-Cys

GSH-adducts UV

DPC

DNA* _

DNA

Replication

DNA-TT

DNA-T6 -- C4

Figure 2 A scheme describing potential mechanisms for the formation of DNA-protein cross-links in mammalian cells. See text for detailed descriptions of the proposed reactions. The abbreviations used are: BSO, L-buthionine sulfoximine; DMSO, dimethylsulfoxide; DNA-ft, DNA containing thymidine dimers; DNA-T6-+C4, DNA containing photoproducts linking C6 and C4 of adjacent pyrimidines; DPC, DNA-protein cross-links; IAA, iodoacetamide; GSH, glutathione; NEM, N-ethylmaleimide.

The significance and repair of DNA-protein cross-links DPC are an order of magnitude less abundant than SSB immediately after irradiation. However, SSB are repaired more rapidly than are DPC, so that after one hour, DPC are in excess of SSB. DPC may be lethal lesions if replication or transcription preceeds repair of DPC in an essential region. It is not yet known if the yield or the repair rate of DPC varies in the cell cycle. In mitotic V79 cells, SSB are repaired, although more slowly than in interphase cells (Oleinick et al., 1984), but DPC produced by y-radiation in metaphase cells are not repaired, even though division appears to be completed. Thus, DPC may be lethal lesions for cells irradiated in metaphase. The mechanism by which DPC are repaired is also not known. When irradiated cell monolayers are incubated at 37°C postirradiation, the level of DPC decreases such that 50% remain after 1 h and less than 10% after 4 h (Chiu et al., 1984, 1986a; Oleinick et al., 1986). The involvement of excision repair is suggested by the accumulation of SSB when normal human fibroblasts were treated with transplatinum, formaldehyde, or potassium chromate and the polymerase step was inhibited with ara-C and hydroxyurea (Fornace, 1982). Studies with the protein synthesis inhibitor, cycloheximide, and with the inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide, have eliminated the possibility of a need for new protein synthesis to replace the cross-linked proteins and suggested the participation of poly(ADP-ribosyl)ation in the second phase of repair of DPC (Chiu et al., 1984). Since DPC are repaired only poorly in human A-549 cells treated with BSO, a requirement for

GSH or an SH-dependent protein is indicated. The inability of metaphase cells to repair DPC implicates the nuclear matrix in the repair mechanism, due to the absence of an intact nuclear matrix in metaphase cells. During repair of DPC, a dynamic interaction between DNA and protein appears to take place (Chiu et al., 1986a; Oleinick et al., 1986). Immediately after irradiation, the DPC are enriched in active DNA. By one hour, the remaining DPC appear to be depleted in those sequences, and by four hours, the enrichment has recovered toward the preirradiation level. The data suggest a hierarchy for repair of DPC. Active DNA cross-linked to nuclear matrix proteins appears to be repaired early, leaving other DNA which does not hybridize to the active DNA probes. Thus, inactive DNA may be repaired late or not at all. One mechanism suggested by the changes in active gene enrichment is a reeling in of sequences to the matrix for repair and the eventual restoration of the initially matrix-bound sequences after repair is complete. The full significance of DPC has yet to be defined. Since the data implicate the nuclear matrix as a preferential site for formation of DPC, it is possible that the conversion of normal non-covalent DNA-matrix associations into tight, covalent structures by free radicals may interfere with normal functions of the nuclear matrix, in particular replication and transcription.

Research in the authors' laboratory is supported by Research Grant CA-15378 from the National Cancer Institute, USPHS, DHHS.

References BANJAR, Z.M., HNILICA, L.S., BRIGGS, R.C., STEIN, J. & STEIN, G.

(1983). Crosslinking of chromosomal proteins to DNA in HeLa cells by UV, gamma radiation and some antitumor drugs. Biochem. Biophys. Res. Commun., 114, 767.

BIAGLOW, J.E., VARNES, M.E., TUTTLE, S.W. & 5 others (1986). The effect of L-buthionine sulfoximine on the aerobic radiation response of A549 human lung carcinoma cells. Int. J. Radiat. Onc. Biol. P/his., 12, 1139.

CHARACTERIZATION OF DNA-PROTEIN CROSS-LINKS CHIU, S.M., OLEINICK, N.L., FRIEDMAN, L.R. & STAMBROOK, P.J.

(1982). Hypersensr.tivity of DNA in transcriptionally active chromatin to ionizing radiation. Biochim. Biophys. Acta, 699, 15. CHIU, S.M., SOKANY, N.M., FRIEDMAN, L.R. & OLEINICK, N.L.

(1984). Differential processing of UV or ionizing radiationinduced DNA-protein cross-links in Chinese hamster cells. Int. J. Radiat. Biol., 46, 681. CHIU, S.M., FRIEDMAN, L.R., SOKANY, N.M., XUE, L.-Y. &

OLEINICK, N.L. (1986a). Nuclear matrix proteins are crosslinked to transcriptionally active gene sequences by ionizing radiation. Radiat. Res., 107, 24. CHIU, S.M., FRIEDMAN, L.R., XUE, L.Y. & OLEINICK, N.L. (1986b). Modification of DNA damage in transcriptionally active vs. bulk chromatin. Int. J. Radiat. Onc. Biol. Phys., 12, 1529. CRESS, A. (1985). Nuclear matrix proteins are covalently linked to DNA after ionizing radiation. Radiat. Res., Abstracts, p. 94. CRESS, A.E. & BOWDEN, G.T. (1983). Covalent DNA-protein crosslinking occurs after hyperthermia and radiation. Radiat. Res., 95, 610. FORNACE, A.J. JR., (1982). Detection of DNA single-strand breaks produced during the repair of damage by DNA-protein crosslinking agents. Cancer Res., 42, 145. FORNACE, A.J. JR. & LITTLE, J.B. (1977). DNA crosslinking induced by X-rays and chemical agents. Biochim. Biophys. Acta, 477, 343. GRUNICKE, H., BOCK, K.W., BECHER, H., GANG, V., SCHNIERDA, J. & PUSCHENDORF, B. (1973). Effect of alkylating antitumor

agents on the binding of DNA to protein. Cancer Res., 33, 1048. HEMMINKI, K. & VAINIO, H. (1979). Preferential binding of benzo[a]pyrene into nuclear matrix fraction. Cancer Lett., 6, 167. MEE, L.K. & ADELSTEIN, S.L. (1979). Radiolysis of chromatin extracted from cultured mammalian cells: Formation of DNAprotein crosslinks. Int. J. Radiat. Biol., 36, 359. MEE, L.K. & ADELSTEIN, S.J. (1981). Predominance of core histones in formation of DNA-protein crosslinks in gamma-irradiated chromatin. Proc. Natl Acad. Sci. (USA), 78, 2194. MEE, L.K. & ADELSTEIN, S.J. (1985). DNA-protein crosslinks in gamma-irradiated chromatin. Proc. of Intl Conference on Mechanisms of DNA Damage and Repair, Gaithersberg, MD. MEYN. R.E., VAN ANKEREN, S.C. & JENKINS, W.T. (1987). The induction of DNA-protein cross-links in hypoxic cells and their possible contribution to cell lethality. Radiat. Res., 109, 419. MILLER, K.G., LIU, L.F. & ENGLUND, P.T. (1981). A homogeneous type II DNA topoisomerase from HeLa cell nuclei. J. Biol. Chem., 256, 9334. MINSKY, B.D. & BRAUN, A. (1977). X-ray-mediated crosslinking of protein and DNA. Radiat. Res., 71, 505.

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NELSON, E.M., TEWEY, K.M. & LIU, L.F. (1984). Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinyl-amino)-methanesulfonm-anisidide. Proc. Natl Acad. Sci. (USA), 81, 1361. NIGGLI, H.J. & CERUTTI, P.A. (1982). Nucleosomal distribution of thymine photodimers following far- and near-ultraviolet irradiation. Biochem. Biophys. Res. Commun., 105, 1215. OLEINICK, N.L., CHIU, S.-M. & FRIEDMAN, L.R. (1984). Gamma radiation as a probe of chromatin structure: Damage to and repair of active chromatin in the metaphase chromosome. Radiat. Res., 98, 629. OLEINICK, N.L., CHIU, S.M., FRIEDMAN, L.R., XUE, L.Y. &

RAMAKRISHNAN, N. (1986). DNA-protein cross-links: New insights into their formation and repair in irradiated mammalian cells. In Mechanisms of DNA Damage and Repair: Implications for Carcinogenesis and Risk Assessment, Simic, M., Grossman, L. & Upton A. (eds) p. 181. Plenum: New York. OLINSKI, R., BRIGGS, R.C., STEIN, J. & STEIN, G. (1983). Crosslinking of chromosomal proteins to DNA in HeLa cells by UV, gamma radiation and some antitumor drugs. Radiat. Res., 86, 102. PEAK, J.G., PEAK, M.J., SIKORSKI, R.S. & JONES, C.A. (1985). Induction of DNA-protein crosslinks in human cells by ultraviolet and visible radiations: Action spectrum. Photochem. Photobiol., 41, 295. RAMAKRISHNAN, N., CHIU, S.M. & OLEINICK, N.L. (1987). Yield of

DNA-protein cross-links in y-irradiated Chinese hamster cells. Cancer Res., 47, 2032. RAYSHELL, M., ROSS, J. & WERBIN, H. (1983). Evidence that Nacetoxy-N-acetyl-2-aminofluorene crosslinks DNA to protein by a free radical mechanism. Carcinogenesis, 4, 501. SMITH, K.C. (1970). A mixed photoproduct of thymine and cysteine: 5-S-cysteine, 6-hydrothymine. Biochem. Biophys. Res. Commun., 39, 1011. SMITH, K.C. (1976). The radiation-induced addition of proteins and other molecules to nucleic acids. In Photochemistry and Photobiology of Nucleic Acids, Wang, S.Y. (ed) 2, p. 187. Academic Press: New York. STRNISTE, G.F. & RALL, S.C. (1976). Induction of stable proteindeoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light. Biochemistry, 15, 1712. WEDRYCHOWSKI, A. WARD, W.S., SCHMIDT, W.N. & HNILICA, L.S.

(1985). Chromium-induced crosslinking of nuclear proteins and DNA. J. Biol. Chem., 11, 7150. WILLIAMS, J:I. & FRIEDBERG, E.C. (1979). Deoxyribonucleic acid excision repair in chromatin after ultraviolet irradiation of human fibroblasts in culture. Biochemistry, 18, 3965.

MULLENDERS, L.H.F., VAN ZEELAND, A.A. & NATARAJAN, A.T.

(1983). Comparison of DNA loop size and super-coiled domain size in human cells. Mutat. Res., 112, 245.

Discussion Michael: Please could you expand on what you alluded to about the role of oxygen. Oleinick: I can't expand a great deal. I can tell you that this is an observation that was made a number of years ago by Al Fornace (A.J. Fornace & J.B. Little (1977), Biochim. Biophys. Acta, 477, 343). He found that there was a larger yield of DNA-protein cross-links as measured by alkaline elution when the cells were irradiated under hypoxic conditions. Ray Meyn (R.E. Meyn et al. (1984), Radiat. Res., 109, 419) has done more experiments on that phenomenon, covering a greater dose range, and has shown that you get a higher ratio of cross-links, to strand breaks under hypoxic conditions. We have done very little more other than to demonstrate with our techniques that we can also see a greater yield in hypoxic cells. I didn't present any of those data, because we really need to do a great deal more with it. The simple explanation would be that a DNA radical reacting with oxygen produces some other damage as opposed to producing a cross-link. In other words, we envision some type of competition. There are a lot of other explanations that could be brought to bear. One of them might be that in the hypoxic cells there is actually a change

in the conformation of DNA that brings different sequences together with protein, and this makes it more amenable to cross-link formation. But until we have done experiments to actually look at the sequences involved in DNA-protein cross-links in hypoxic cells we can't draw any conclusions.

Michael: Were all the measurements and values you showed obtained for cells cultured anaerobically or in oxygen? Oleinick: Everything we have done is with aerobic cells. There is a yield of cross-links in oxygen, but there is a greater yield under hypoxia, but I haven't shown those data. Cramp: What fraction of the total protein of the nucleus do you get withheld on your filters? Oleinick: That depends - of the nucleus, at least half of it. There is a trcmecedous amiiount of protein autached to the filter. Cramp: What is your lysis procedure? Oleinick. We lyse the cells in one 1 M sodium perchlorate