The repair of DNA damage in eukaryotic cells is closely coupled with local changes of chromatin structure such that newly synthesized repair patches transiently appear in ‘free' DNA domains with increased accessibility to enzymatic and chemical probes. We have isolated these domains from mammalian cells repairing bulky DNA adducts. During the first 3 h of repair, excision of adducts occurred exclusively in free DNA and was closely linked with the appearance of newly synthesized repair patches. Following depletion of chromatin-bound poly(ADP-ribose), the repositioning of repair patches into these domains was completely blocked, although overall repair patch synthesis was unaltered. Concomitantly, DNA adducts were no longer excised and tended to accumulate in free DNA domains. Our results suggest a tight coupling of the excision step with the formation of free DNA domains by a mechanism involving poly ADP-ribosylation of chromatin protein

Althaus, Felix R.

Mathis, Georg

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Carcinogenesisvol.il no.7 pp.1237-1239, 1990SHORT COMMUNICATIONUimcoiuipMinig of BNA excisionpo!y(AID)P-ribaise)-dlepIeftedl mm;and inundeosoinnijcellsig mGeorg Mathis and Felix R.Althaus1University of Zurich (Tierspital), Institute of Pharmacology andBiochemistry, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland'To whom correspondence should be addressedThe repair of DNA damage in eukaryotic cells is closelycoupled with local changes of chromatin structure such thatnewly synthesized repair patches transiently appear in 'free'DNA domains with increased accessibility to enzymatic andchemical probes. We have isolated these domains frommammalian cells repairing bulky DNA adducts. During thefirst 3 h of repair, excision of adducts occurred exclusivelyin free DNA and was closely linked with the appearance ofnewly synthesized repair patches. Following depletion ofchromatin-bound poly(ADP-ribose), the repositioning ofrepair patches into these domains was completely blocked,although overall repair patch synthesis was unaltered.Concomitantly, DNA adducts were no longer excised andtended to accumulate in free DNA domains. Our resultssuggest a tight coupling of the excision step with the formationof free DNA domains by a mechanism involving poly ADP-rnbosylation of chromatin proteins.In chromatin of mammalian cells, newly synthesized DNA repairpatches exhibit a transient micrococcal nuclease hypersensitivity.This hypersensitivity is thought to reflect local disruptions in thetightly packed nucleosomal organization of chromatin, causingexposure of 'free' DNA domains (for review see 1,2). Thefunction of these domains as well as the mechanisms involvedin their formation are unknown. We have speculated that thepost-translational poly ADP-ribosylation of chromatin proteinsmight be involved in the formation of free DNA domains in DNAexcision repair. Poly ADP-ribosylation is catalyzed by the enzymepoly(ADP-ribose)polymerase (1,3,4; EC 2.4.2.30). Followingactivation by DNA nicks, this enzyme operates in a strictlyprocessive manner (5). Continuous treatment of living cells withbenzamide, a competitive inhibitor of this enzyme (3,4,6), resultsin the degradation of chromatin-bound ADP-ribose polymers (7)by the enzyme poly(ADP-ribose)glycohydrolase (3,4). Usingnon-replicating adult rat hepatocytes in primary monolayerculture, we have established conditions for the complete depletionof chromatin-associated poly(ADP-ribose) (7,8). Hepatocytessurvive up to 9 days under these conditions and maintainexpression of liver-specific functions (8). Thus, poly(ADP-ribose)-depleted hepatocytes represent a convenient model systemto study the role of poly ADP-ribosylation in specific steps ofDNA repair.We have previously shown that unfolded free DNA domainscan be isolated from the chromatin of intact mammaliancells by taking advantage of their preferential accessibility to8-methoxypsoralen (9). Upon intercalation into free DNAdomains of living cells, 8-methoxypsoralen can be photoactivatedto form bifunctional DNA adducts crosslinking the two DNAstrands. Crosslinked (free) DNA domains can then be isolatedquantitatively following a denaturation/renaturation treatment andsubsequent nuclease SI digestion of non-crosslinked DNA strands(for details see 9).Here we have isolated free DNA domains from non-replicatinghepatocytes (9), which had been induced to repair bulky DNAadducts by treatment with a low dose of the ultimate carcinogenA/-acetoxy-2-acetylaminofluorene. Figure 1 shows that newlysynthesized repair patches, pulse-labeled for an initial 20 minrepair period, gradually accumulate in free DNA domains duringsubsequent chase periods. This observation is in accordance withprevious results based on nuclease probing of chromatin (1,2),or direct isolation of these domains (9).No repositioning of repair patches into free DNA domains wasobserved in poly(ADP-ribose)-depleted hepatocytes (7; Figure1, legend). This depletion did not affect the overall synthesis ofrepair patches (Figure 1, legend). Thus, the synthesis andrepositioning of repair patches in poly(ADP-ribose)-depletedchromatin of hepatocytes were uncoupled.Figure 2 provides a comparison of excision activity in totalchromatin (Figure 2A) and free DNA (Figure 2B) of poly(ADP-ribose)-depleted and undepleted hepatocytes. In the first 3 h ofProtocolPulse: 0Chase: 20Chase: 120•»20t»120t»180minminminRedistribution of RepairPatchesi 'ii i—i\ \• i i iFig. 1. Redistribution of repair patches relative to free DNA domains inpoly(ADP-ribose)-depleted and undepleted hepatocytes. Parenchymal livercells were isolated from adult rat hepatocytes and cultured for 24 h in thepresence or absence of 8 mM benzamide as previously described (9,31).The level of chromatin-bound poly(ADP-ribose) in benzamide-treated cellswas < 10 fmol ADP-ribosyl residues/106 cells, as determined by the methodof Jacobson and co-workers (32,33). Hepatocytes were then exposed to50 /M yV-acetoxy-2-acetylaminofluorene (NCI Chemical CarcinogenRepository, Bethesda, MD, USA) and newly synthesized repair patcheswere pulse-labeled with [methyl-3H]thymidine (49 Ci/mmol, 20 /tCi/3 mlmedium) for 20 min. Incorporation of radioactivity was stopped by twochanges of medium containing 1.2 fiM unlabeled thymidine, followed by achase in the continuous presence or absence of benzamide. At the end ofthese incubations, free DNA domains were isolated from hepatocellularchromatin as previously described (9) and analyzed for their content ofrepair patches (9). The numbers plotted on the abscissa represent the ratiosof radioactivity (d.p.m.//ig DNA) incorporated into free DNA to theradioactivity incorporated into bulk DNA. The values reflect the relativeaccumulation of repair patches in free DNA (9) (a value of 1 indicates noaccumulation) and represent the mean ± SEM of four independentexperiments with separate cell preparations. The overall repair incorporationwas 511.2 ± 85.4 mmol (controls) and 408.2 ± 94.8 mmol (depleted cells)[methyl-3H]thymidine/mol adduct formed (mean ± SEM, n = 3). Blackbars: control cells; shaded bars: poly(ADP-ribose)-depleted cells.Oxford University Press 1237G.Mathis and F.R.AIthaus100-~ 75-100-o<0 20 120Repair Time (min)Fig. 2. Excision of deoxyguanosine adducts in total DNA and free DNAdomains of poly(ADP-ribose)-depleted and undepleted hepatocytes exposedto A/-acetoxy-2-acetylaminofluorene (AAAF). Hepatocytes were incubated for20 min with [G-3H]AAAF (232 mCi/mmol, total concentration 50 ^M) andthe amount of deoxyguanosine adducts was quantified following DNApurification (9). (A) Adduct removal in total DNA; (B) adduct removal infree DNA domains. O O, poly(ADP-ribose)-depleted cells; O O,undepleted control cells. The results represent the means ± SEM of threeindependent experiments involving separate cell preparations.0 20 120Repair Time (min)180Fig. 3. Relative distribution of the remaining adducts in free DNA domainsof poly(ADP-ribose)-depleted and undepleted hepatocytes after various repairintervals. Hepatocytes were incubated with radiolabeled A'-acetoxy-2-acetyl-aminofluorene as described in Figure 2 and the deoxyguanosine adductconcentration was quantified in free DNA and expressed relative to theircontent in total DNA. O O, poly(ADP-ribose)-depleted cells; O O,undepleted cells. The values plotted on the ordinate represent the ratios of[G-3H]AAAF radioactivity in free DNA to the radioactivity contained inbulk DNA. The results represent the means ± SEM of three independentexperiments involving separate cell preparations.repair, the rate of excision of deoxyguanosine adducts formedduring a 20 min incubation of hepatocytes with /V-acetoxy-2-acetylaminofluorene was almost identical in total chromatin andfree DNA. However, no excision of these adducts occurredduring the same time period in poly(ADP-ribose)-depleted cells(Figure 2). Hence the repositioning of repair patches (Figure 1)seems to be coupled with adduct excision (Figure 2) by a mech-anism involving de novo poly ADP-ribosylation of chromatinproteins. This is compatible with the idea that unfolded DNAdomains are a preferential site of excision activity (Figure 2).The results in Figure 3 suggest that some unfolding ofchromatin occurs also in poly(ADP-ribose)-depleted cells. Aslight accumulation of unexcised DNA adducts in the free DNAfraction was obtained in poly(ADP-ribose)-depleted cells, whileno significant accumulation was seen in undepleted control cells.Thus, the unfolded domains formed in poly(ADP-ribose)-depletedcells are enriched in DNA adducts and largely deficient in repairpatches (cf. Figures 1 and 3).Our results thus reveal a tight functional linkage betweenstructural chromatin rearrangements and the excision step, anddissect the process of repair patch synthesis from the patchrepositioning relative to nucleosomally organized chromatinregions. Moreover, our results tentatively identify a requirementfor poly ADP-ribosylation in co-ordinating excision repair withstructural rearrangements of chromatin. The role of this post-translational protein modification is poorly understood, but allprotein acceptors of poly(ADP-ribose) hitherto identified (forreview see 4) share the capacity to bind to nucleic acids. PolyADP-ribosylation of these proteins in vitro reversibly alters theirDNA binding affinity (10-15). Enzymatic addition and removalof ADP-ribose polymers on chromatin preparations in vitroinduces reversible relaxation of polynucleosomes (16), therelaxation state being directly related to the size of histone-boundADP-ribosyl polymers (17-19). Histones are also a physiologicaltarget of ADP-ribose modifications in carcinogen-treatedmammalian cells in vivo (20,21). Thus, the reversiblemodification of DNA-protein interactions (14,15,22,23) byprotein ADP-ribosylation may be the underlying molecularmechanism for the transient formation of free DNA domains ineukaryotic excision repair.As far as our findings ascribe poly ADP-ribosylation a rolein DNA excision repair, they contrast the hypothesis of Shalland associates who envisioned poly ADP-ribosylation as adirect regulatory mechanism of ligation activity in the repair ofalkylation damage in DNA (24,25). However, other reports couldnot confirm such a role for poly(ADP-ribose) in DNA excisionrepair (26-30). This contradictory phenomenology may bereconciled on the premise that poly ADP-ribosylation affects localdisruptions of chromatin structure and this secondarily affectsDNA repair reactions (4,26-30).AcknowledgementsWe thank Phyllis Panzeter for critically reviewing this manuscript. This workwas supported by grant 3.161.088 from the Swiss National Foundation forScientific Research, and the Jubilaumsstiftung of the University of Zurich.Referencesl.Friedberg.E.C. (1985) DNA Repair. W.H.Freeman, New York.2.Smerdon,M.J. (1990) In Lambert.M.W. and Laval.J. (eds), DNA RepairMechanisms and their Biological Implications in Mammalian Cells. Plenum,New York, pp. in press.3. Ueda,K. and Hayaishi,O. (1985) ADP-ribosylation. Annu. Rev. Biochem.,54, 73-100.4. Althaus.F.R. and Richter,C. (1987) Molecular Biology, Biochemistry, andBiophysics, Vol. 37: ADP-ribosylation of Proteins: Enzymology and BiologicalSignificance. Springer-Verlag, Berlin.5. Naegeli,H. and Althaus,F.R. (1989) Poly ADP-ribosylation of proteins:processivity of a posttranslational modification. J. Biol. Chem 26414382-14385.6. Rankin,P.W., Jacobson.E.L., Benjamin,R.C., Moss,J. and Jacobson,M.K.(1989) Quantitative studies of inhibitors of ADP-ribosylation in vitro andin vivo. J. Biol. Chem., 264, 4312-4317.7. Alvarez-Gonzalez,R. and Althaus.F.R. (1989) Poly(ADP-ribose)catabolismin mammalian cells exposed to DNA-damaging agents. Mutat. Res., 21867-74.8. Althaus.F.R., Lawrence,S.D., He,Y.Z., Sattler.G.L., Tsukada,Y. andPitot,H.C. (1982) Effects of altered [ADP-ribose]n metabolism on expressionof fetal functions by adult rat hepatocytes. Nature, 300, 366-368.9. Mathis,G. and Althaus.F.R. (1986) Periodic changes of chromatin organizationassociated with rearrangement of repair patches accompanying DNA excisionrepair of mammalian cells. J. Biol. Chem., 261, 5758-5765.10. Gaal,J.C. and Pearson,C.K. (1985) Eukaryotic nuclear ADP-ribosylationreactions. Biochem. 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Uncoupling of DNA excision repair and nucleosomal unfolding in poly (ADP-ribose)-depleted mammalian cells

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