Rapid DNA–Protein Cross-Linking and Strand Scission by an Abasic Site in a Nucleosome Core Particle

Rapid DNA–protein cross-linking and strand scission by an abasic site in a nucleosome core particle

Jonathan T. Sczepanskia, Remus S. Wonga, Jeffrey N. McKnightb, Gregory D. Bowmanb, and Marc M. Greenberga,1

aDepartment of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218; and bDepartment of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218

Edited by Peter B. Dervan, California Institute of Technology, Pasadena, CA, and approved November 12, 2010 (received for review August 30, 2010)

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are within ∼1.5 turns of the DNA duplex, are an important family of highly mutagenic and cytotoxic if not repaired. In addition, clusters lesions produced in DNA exposed to γ-radiolysis. Bistranded of two or more abasic lesions within one to two turns of DNA, a clustered lesions are repaired much less efficiently than the re- hallmark of ionizing radiation, are repaired much less efficiently spective isolated lesions by members of the base excision repair and thus present greater mutagenic potential. Abasic sites are che- (BER) pathway. For example, a clustered lesion comprised of two mically labile, but naked DNA containing them undergoes strand AP sites on opposite strands that are displaced 5′ to one another scission slowly with a half-life on the order of weeks. We find that significantly resists Ape1 incision in vitro (9). Moreover, AP sites independently generated AP sites within nucleosome core particles that are part of clustered lesions persist in cells even after 1 d, are highly destabilized, with strand scission occurring ∼60-fold whereas isolated lesions are repaired in 1 h (10, 11). The lifetimes more rapidly than in naked DNA. The majority of core particles of lesions in DNA are also affected by their rotational orientation containing single AP lesions accumulate DNA–protein cross-links, within the nucleosome, which can limit their accessibility to repair which persist following strand scission. The N-terminal region of proteins (12, 13). For electrophilic lesions such as AP, this en- histone protein H4 contributes significantly to DNA–protein cross- hances the possibility that chemical reactions will occur with neigh- links and strand scission when AP sites are produced approximately boring nucleobases and/or protein residues to form DNA–DNA 1.5 helical turns from the nucleosome dyad, which is a known hot interstrand cross-links (14–16) and DNA–protein cross-links spot for nucleosomal DNA damage. Reaction rates for AP sites at (DPCs), respectively (17, 18). DPCs require alternative repair BIOCHEMISTRY two positions within this region differ by ∼4-fold. However, the pathways from BER (19–21). In addition, one DNA interstrand strand scission of the slowest reacting AP site is accelerated when cross-link arising from an abasic lesion is misrepaired by nucleo- it is part of a repair resistant bistranded lesion composed of two AP tide excision repair and transformed into an even more deleterious sites, resulting in rapid formation of double strand breaks in high double strand break (22). Abasic sites are also prone to elimina- yields. Multiple lysine residues within a single H4 protein catalyze tion, which results in strand scission and the release of an even double strand cleavage through a mechanism believed to involve a more electrophilic α,β-unsaturated aldehyde (23, 24). templating effect. These results show that AP sites within the nu- DNA of eukaryotic genomes is packaged into arrays of nucleo- cleosome produce significant amounts of DNA–protein cross-links somes that comprise chromatin structure. The nucleosome core CHEMISTRY and generate double strand breaks, the most deleterious form of particle consists of 146–147 bp of DNA wrapped around a histone DNA damage. protein octamer containing two copies of four histone proteins (H2A, H2B, H3, and H4) (25). The lysine-rich N-terminal tails chromatin ∣ oxidative damage ∣ histone modification of the histones extend from the protein core, making various con- tacts with the DNA minor groove. The proximity of nucleosomal xogenously and endogenously produced agents continuously AP sites to these basic residues is expected to accelerate cleavage Edamage DNA. More than 70 types of damage have been iden- of the lesion relative to naked DNA (26–28). Previously, Povirk tified, and significant effort is expended to determine which of showed that the oxidized abasic lesion produced by bleomycin these lesions are biologically important (1, 2). The apurinic/apyr- undergoes rapid strand scission when produced in chromatin imidinic (AP) site is a ubiquitous form of DNA damage that (27). However, this reaction, and the behavior of nucleosomal is produced in excess of 10,000 lesions per cell each day (3). This abasic sites in general, are not well characterized. Therefore, highly mutagenic lesion results from spontaneous hydrolysis of we prepared nucleosome core particles containing precisely posi- native and damaged nucleotides. AP sites are also formed as in- tioned AP sites using a stable photolabile precursor (4, Fig. 1C), termediates during base excision repair of alkylated and oxidized which allowed for the complete generation of AP within 10 min. nucleotides and are themselves removed by multiple enzymatic We find that isolated AP lesions in the nucleosome are at least 60 pathways (4, 5). In mammalian cells incision of the AP’s5′-phos- times more reactive than in naked DNA of the same sequence. – phate by Ape1, followed by excision of the resulting 5′-terminal This reaction involves formation of slowly reversible DNA pro- 2′-deoxyribose phosphate by DNA polymerase β is the major tein cross-links believed to involve Schiff base formation between pathway for removing the lesion. Redundant repair pathways are AP sites and histone lysine residues. The enhanced lability of AP reflective of the high mutagenic potential and large quantities of sites results in double strand breaks (DSBs) when they are part AP produced and are indicative of their physiological significance. of a clustered lesion that resists BER. Importantly, the rate of Mammalian cells lacking Ape1, the major base excision repair double strand cleavage is faster than that expected based upon protein responsible for incising AP sites, are embryonic lethal (6). the reaction of two, isolated AP sites at the same positions. These Recent observations reinforce the perception that AP sites are

cytotoxic. For instance, formation of bursts of AP sites by the Author contributions: J.T.S. and M.M.G. designed research; J.T.S. and R.S.W. performed natural product leinamycin is postulated to be the source of this research; J.N.M. and G.D.B. contributed new reagents/analytic tools; J.T.S., R.S.W., and antitumor agent’s cytotoxicity (7). In addition, increasing the life- M.M.G. analyzed data; and J.T.S., G.D.B., and M.M.G. wrote the paper. time of AP sites in cells via the expression of an inactive form of The authors declare no conflict of interest. Ape1 enhances the cytotoxicity of DNA damaging drugs (8). This article is a PNAS Direct Submission. Prolonging the lifetime of DNA lesions increases the likeli- 1To whom correspondence should be addressed. E-mail: [email protected]. hood that they will be present during replication, resulting in mu- This article contains supporting information online at www.pnas.org/lookup/suppl/ tations. Clustered lesions, consisting of two or more damage sites doi:10.1073/pnas.1012860108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1012860108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Independent generation of AP sites within nucleosome core particles. (A) X-ray crystal structure of the α-satellite DNA nucleosome core particle (PDB ID code 1aoi) highlighting positions of AP incorporation. (B) A portion of the α-satellite DNA sequence showing where the AP sites are incorporated (see SI Text for complete DNA sequences). (C) Methods used for independently generating AP sites.

data suggest Schiff base formation with one AP site accelerates ples by SDS PAGE, which dehybridizes noncovalently bound reaction with a second lesion by acting as a template and increas- DNA–protein complexes, allowed for simultaneous quantifica- ing the effective molarity of a lysine side chain(s) in the vicinity of tion of intact DNA within core particles, DNA containing single the second lesion. Moreover, these experiments reveal isolated strand breaks (SSBs) at the AP site, and DPCs. However, it does AP lesions are efficient sources of DNA–protein cross-links, not distinguish DPCs containing a SSB from those in which the and that bistranded AP lesions, such as those produced by ioniz- DNA is intact. To determine total strand scission regardless of ing radiation, are substantial sources of double strand breaks in the presence of DPCs, nucleosome core particles were digested nucleosomal DNA. with proteinase K and the DNA separated by denaturing PAGE. All samples were treated with the reducing agent NaBH4 prior to Results electrophoretic analysis, which minimized subsequent adventi- Preparation of Nucleosome Core Particles Containing Site-Specific AP tious cleavage of AP. The reducing agent also trapped any DPCs α Sites. The -satellite palindrome sequence was chosen as a host resulting from Schiff base formation. In situ trapping of Schiff for the AP lesion due to its well characterized structure (25, 29). base intermediates was accomplished by incubating samples in In particular, the region near superhelical location (SHL) 1.5 the presence of NaBH3CN. (AP89,AP207) was chosen for AP incorporation because it is re- Approximately 20% of intact, unmodified DNA remained in cognized as a hot spot for drug binding (Fig. 1A and B) (30, 31). nucleosome core particle 1a after a 32-h incubation under phy- This region is significantly bent and is in close proximity to the siological conditions (pH 7.5, 37 °C) (Fig. 3A). The majority of lysine-rich tails of histones H3 and H4. AP sites at positions DNA in 1a (∼68%) was cross-linked to a histone protein(s). 89 and 207 were generated via 10-min photolysis using long Denaturing PAGE analysis of proteinase K digested 1a revealed wavelength light (350 nm) of nucleosome core particle or naked 4 C 4 that >80% of AP89 (Fig. 1B) had undergone elimination during DNA containing (Fig. 1 ). Short oligonucleotides containing B (Scheme S1) were prepared via solid phase chemical synthesis, the incubation (Fig. 3 ), indicating that a significant amount and the 146-bp α-satellite DNA duplexes were constructed by of the DNA in the DPCs was cleaved. This was corroborated ligating these with two longer biopolymers that were also chemi- cally synthesized (Fig. S1). To ensure selective 5′-32P labeling of the strand containing 4, the DNA substrates were constructed such that the strand opposite the lesion was blocked synthetically at its 5′ end. The substrates containing the clustered AP lesion (3a–3d) were constructed such that either strand could be monitored independently using 32P. All 146-bp duplexes were purified by native PAGE, and nucleosome core particles were reconstituted via stepwise salt reduction using recombinant Xenopus laevis histone proteins expressed in Escherichia coli (32). Nucleo- some reconstitutions containing less than 15% free DNA were used without further purification, and all values reported herein have been corrected to account for any remaining free DNA. The rotational orientation of each nucleosome core particle containing AP precursor 4 was determined through DNase I digestion (Fig. S2), which confirmed proper positioning of all substrates with respect to the X-ray structure (33, 34). Completeness of AP generation from 4 was confirmed by PAGE analysis, following alkaline treatment (Fig. S3). Photochemical generation of an AP site is advantageous over the commonly used hydrolysis of 2′-deoxyuridine (dU) by uracil DNA glycosylase (Fig. 1C) because the enzyme is inhibited by the presence of a nucleosome Fig. 2. Strategy for using gel electrophoresis to analyze the reactivity of AP and its activity depends upon the rotational orientation of dU in nucleosome core particles. (Top) Expected cross-linking and cleavage reac- within the core particle (12, 13). Rapid generation of AP inde- tions between an AP site and a lysine residue. (Bottom) Predicted detection pendent of its position within the core particle was particularly of different products using two electrophoresis methods. Denaturing PAGE following histone digestion by proteinase K separates intact nucleosomal helpful when following the time course of reactions. DNA (black) from cleaved DNA (red) whether or not the DNA was cross-linked to protein. SDS PAGE separates intact nucleosomal DNA (black), AP Lesions Display Accelerated Reactivity in Nucleosome Core Parti- DNA–protein cross-links (DPC, blue), and cleaved DNA that is not cross-linked cles. The reactivity of AP within nucleosome core particles was to protein (SSB, green). Please note that SDS PAGE does not distinguish monitored via various forms of PAGE (Fig. 2). Analysis of sam- between DPCs containing cleaved and intact DNA (blue).

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1012860108 Sczepanski et al. Downloaded by guest on September 24, 2021 Clustered AP Lesions in the Nucleosome Accelerate Formation of DNA Double Stranded Breaks. The reactivity of nucleosome core particles containing bistranded AP lesions displaced 5′ to one another by 3 nucleotides (3a, 3c) were examined (Fig. 4 and Fig. S6). The position of the AP sites in the nucleosomal DNA (89, 207) of these core particles are the same as in those containing isolated lesions (1, 2) and differ from one another only with respect to which strand of the DNA is radiolabeled (Fig. 1B). This spatial distribution of lesions was chosen because it significantly inhibits repair and can be expected to have a long lifetime in cellular DNA (9, 35). The rate constant for the disappearance of intact Fig. 3. Reaction of AP89 in a nucleosome core particle (1a) and naked DNA of 3a 3c the same sequence. (A) SDS PAGE analysis describing disappearance of intact DNA in core particles and (also fit to first order) was es- DNA containing AP89 in nucleosome core particle 1a and growth of DPC and sentially the same regardless of which strand was labeled k ∼ 2 6 × 10−5 −1 SSB as a function of time. (B) Comparison of the total amount of strand ( Dis . s ) Furthermore, denaturing PAGE analysis scission using denaturing PAGE analysis in naked, and nucleosomal DNA from of 3a and 3c following proteinase K digestion revealed that 1a, following protease digestion. AP89 and AP207 underwent elimination at rates that were indis- tinguishable from one another when part of the bistranded lesion by subsequently analyzing the DNA from isolated DPCs by de- (Fig. S7), which is consistent with the lack of significant SSB ac- naturing PAGE, which revealed ∼50% of the cross-linked DNA cumulation in these substrates (Fig. 4). This suggests AP207 reacts was cleaved within 1 h and that at least 90% was cleaved at any ∼4-fold faster when it is part of a clustered lesion (3c) than when time after 17 h (Fig. S4). The product mixture was simplified by it is isolated in the core particle (2a). Importantly, naked DNA incubating core particle in the presence of NaBH3CN (10 mM). containing the bistranded lesion was not any more reactive than This resulted in a modest increase in overall DPC formation that containing a single AP site (Fig. S3). Although the reactivity (∼85% after 32 h, Fig. S5) and a significant reduction of cleaved of AP207 increased in the bistranded lesion, DNA–protein cross- DNA in the DPCs (∼20%)(Fig. S4). These data strongly suggest link growth and stability were lower compared to that in nucleo- that cleavage at AP89 is dependent on formation of a Schiff base some core particles containing an isolated AP site (Fig. 4A). and that hydrolysis of this intermediate following AP elimination Removal of the lysine-rich H4 tail also reduced the reactivity – 3b k ¼ is slow. Finally, despite rapid DNA protein cross-link formation, of AP89 and AP207 in the bistranded complexes ( : Dis BIOCHEMISTRY 1 1 × 10−5 −1 3d k ¼ 8 5 × 10−6 −1 no DNA interstrand cross-links were detected in any of these . s ; : Dis . s )(Fig. S6) to a similar substrates (14). extent (∼2.5-fold) as was observed in core particles containing The kinetics for disappearance of AP sites in nucleosome core isolated AP lesions (Table 1). particles 1a and 2a were approximated by ignoring the reversibil- Enhanced AP site reactivity in the nucleosome core particle ity of initial Schiff base formation, and fitting the data to a first- containing a clustered lesion gave rise to rapid double strand order reaction (Table 1). The half-life of AP89 within the nucleo- break formation (Fig. 4). Some double strand breaks are dis- some was at least 60-fold shorter than in naked DNA (24). So guised by cross-linking to histone protein(s), but treatment of ali- CHEMISTRY little strand scission was observed in the naked DNA used to con- quots with proteinase K unmasked all strand scission (Fig. 4B). struct nucleosome core particle 1a under identical conditions Double strand break formation proceeded rapidly after a short, B k ∼ 3 9 × 10−7 −1 (Fig. 3 ) that the estimated rate constant ( Dis . s , barely discernible induction period between 0 and 6 h. More than t1∕2 ∼ 500 h) is likely a maximum, and the 60-fold acceleration of 30% of the DNA underwent double strand cleavage in 6 h and AP89 reactivity in the region of SHL 1.5 within the α-satellite nu- double strand breaks reached more than 90% after incubating cleosome core particle is a minimum. AP was also destabilized in the nucleosome core particle (3a) for 32 h. Analysis of an aliquot nucleosomes when introduced three nucleotides away on the removed after 32 h by native PAGE did not show any decomposi- opposite strand (AP207, 2a) (Table 1). The variation in reactivity tion of the core particle (Fig. S6), indicating that double strand between these two positions (∼4-fold) was small compared to the break formation did not result in nucleosome decomposition. overall acceleration experienced in the nucleosome core particle. Removing the lysine-rich tail of H4 reduced the efficiency of Structural data suggest the lysine-rich N-terminal tail of his- double strand break formation to a similar extent as it did the tone H4 is within proximity to the DNA at SHL 1.5 (25, 29). reaction of isolated AP lesions (Fig. S6). Therefore, core particles (1b, 2b) lacking 19 residues of the H4 tail (amino acids 1–19) were examined in order to determine Identification of Histone Proteins Involved in AP Cross-Linking. Schiff what role (if any) the tail plays in the enhanced reactivity of AP at base formation between AP sites and lysine residues were this location. In general, the reactivity of both AP sites at SHL 1.5 assumed to account for the accumulation of large amounts of decreased in core particles lacking the H4 tail (Table 1). For in- k stance, the rate constant for AP89 disappearance ( Dis) decreased more than 3-fold when the reconstituted core particle contained a tailless H4 mutant (1b)(Fig. S5). The effect of removing the H4 tail on DPC formation correlated with the reduction noted in AP89 elimination (Fig. S5). The effect of removing the histone H4 tail was somewhat smaller on the reactivity of AP207. The rate constant describing AP207 disappearance decreased less than 2-fold when the H4 tail was removed (2b).

Table 1. Reactivity of AP sites in nucleosome core particles k ×10−6 −1 t Substrate Position H4 tail Dis, s 1∕2 AP, h Fig. 4. Reaction of a bistranded lesion (AP89,AP207) in a nucleosome core particle (3a). (A) Disappearance of intact core particle 3a and growth of 1a AP89 + 22.7 ± 3.5 8.5 DPC, SSB, and DSB as a function of time using SDS PAGE (without proteinase 1b AP89 − 7.5 ± 1.4 25.7 K treatment of aliquots). (B) Time dependence of the disappearance of intact 2a AP207 + 6.1 ± 0.7 31.6 nucleosome core particle 3a, and the growth of products, using SDS PAGE 2b AP207 − 3.7 ± 0.6 52.0 following proteinase K digestion of aliquots.

Sczepanski et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 24, 2021 Fig. 6. Identification of the histone proteins involved in DPC formation with AP sites in the bistranded lesion. (A) DPC formation with AP89 (nucleosome Scheme 1. Method for determining protein(s) cross-linked with AP sites. core particle 3a, 3b) and AP207 (nucleosome core particle 3c, 3d)inthe presence of wild-type (3a, 3c) and tailless H4 protein (3b, 3d). (B) Overall histone–DNA cross-links during nucleosome core particle pro- DNA–protein cross-link yield, and contribution by AP89 (3a) and AP207 (3c)to moted DNA cleavage. In order to identify the protein(s) involved DPCs in the bistranded substrate when incubated in the presence of NaBH3CN. Individual contributions by AP89 and AP207 were determined by in cross-linking, we devised an assay that involved cross-link denaturing samples prior to gel electrophoresis. dependent transfer of the 32P-radiolabel from the DNA to the corresponding protein (Scheme 1) (Fig. S4). Core particles comprised of DNA containing 5′-32P-AP sites were incubated in the compared to ∼8% when the bistranded lesion was part of a nu- 3c presence of NaBH3CN in order to trap DPCs involving a Schiff cleosome core particle composed of all wild-type proteins ( ). base at the site of the radiolabel. The DNA was subsequently Incubation of 3a (and 3c) was carried out in the presence of digested with DNase I and nuclease P1, which leaves a short NaBH3CN in order to determine if simultaneous cross-linking of DNA fragment(s) containing the reduced, cross-linked 5′-32P-AP both AP sites to different residues of histone H4 was possible. site bonded to the histone. The histone proteins were separated The overall amount of DPCs in 3a (the strand containing by SDS and Triton/Acid/Urea (TAU) PAGE and visualized with AP89 is labeled) increased significantly in the presence of the re- Coomassie blue staining, as well as by autoradiography (Fig. S4). ducing agent (∼93%, Fig. 6B). Importantly, the level of DPCs was Histone H4 was the major (>95%) protein trapped upon incuba- unaffected when the DNA was denatured prior to analysis by SDS tion of nucleosome core particles containing AP89 whether (1a) PAGE, indicating that every cross-linked duplex consisted of a or not (1b) the lysine-rich tail of H4 was present (Fig. 5A and covalent bond between AP89 and a histone. The level of DPCs Fig. S4). A relatively modest translocation of the AP site 3 nucleo- to the strand containing AP207 (using nucleosome core particle tides upstream on the opposite strand of the duplex (AP207, nu- 3c) was slightly lower under similar conditions. However, the cleosome core particle 2a) resulted in a slight decrease in cross- summation of DPCs involving AP89 and AP207 under denaturing linking to H4 and a commensurate increase in H2A (+4%) and conditions was far greater than the overall amount of cross-linked H3 (+2%) participation. However, removing the lysine-rich tail duplex. Given that the majority of DPCs at AP89 and AP207 in- from H4 (2b) significantly reduced the amount of cross-linking volve histone H4 (Fig. 6A), this indicates that both strands were between AP207 and this protein, which was compensated for by simultaneously cross-linked to that protein in a significant portion an increase in trapping by H3 but not H2A or H2B (Fig. 5B). of core particles. The distribution of histone proteins involved in cross-linking of the bistranded lesion was very similar to those that react with Discussion isolated AP sites. Cross-linking to the individual AP sites was de- The mutagenicity of AP sites and the biological importance of termined by generating the appropriate 5′-32P-AP in the top (3a, their efficient repair are widely appreciated (4). More recently, 3b,AP89) or bottom (3c, 3d,AP207) strand of the DNA within the AP lesions were shown to form interstrand cross-links within nucleosome core particle (Fig. 1B). Overall, the majority of DPCs DNA, raising the possibility that potentially more deleterious le- (>85%) involved histone H4, regardless of the AP site being mea- sions could be produced from them (14). In this work we report sured (Fig. 6A). Together, histones H4 and H3 account for more on the reactivity of individual AP lesions that were independently than 95% of the DPCs in wild-type core particles containing the generated in a region of nucleosome core particles (SHL 1.5) that bistranded lesion. The contribution of H3 to cross-linking AP89 in is a hot spot for DNA damage (30, 31). Although DNA–DNA 3a was ∼11% compared to ∼3% in the substrate containing the cross-links were not detected when isolated AP sites were inde- isolated lesion (1a). Furthermore, the amount of cross-linking to pendently produced at defined sites in core particles, we observed H3 increased to more than 15% when the H4 tail was removed an ∼60-fold increase in AP site reactivity within the nucleosome (3b). The effect of the H4 tail on cross-linking to AP207 was con- compared to free DNA, resulting in a reduction of the lesion’s siderably larger in the bistranded lesion (3c, 3d). Cross-linking to half-life to hours (24). Isolated AP lesions rapidly led to DNA– H3 increased to more than 35% when the H4 tail was absent (3d), protein cross-links and strand breaks. In addition, large amounts of DPCs persisted after AP elimination. In general, less is known about DPC repair than of other types of DNA lesions (21, 36). Even less is known about DPC repair in nucleosomes and how these lesions affect chromatin remodeling. The observations reported above suggest that site-specific incorporation of AP sites may be a good way of producing such systems for further study. Kinetic and trapping experiments conducted on nucleosome core particles composed of wild-type or a mutant form of histone H4 lacking its lysine-rich tail (residues 1–19) show that this portion of the protein contributes to the accelerated cleavage Fig. 5. Identification of the histone proteins involved in DPC formation with of AP sites in the vicinity of SHL 1.5. Removal of the H4 tail AP sites in nucleosome core particles. (A) DPC formation with AP89 in the presence of wild-type (nucleosome core particle 1a) and tailless H4 protein results in only modest reduction in AP site cleavage compared (nucleosome core particle 1b). (B) DPC formation with AP207 in the presence to core particle comprised of wild-type histones. Furthermore, of wild-type (nucleosome core particle 2a) and tailless H4 protein (nucleo- despite lacking 19 amino acid tail residues, the mutant H4 protein some core particle 2b). See also Fig. S4. remains the major contributor to DPCs at AP89 and AP207. These

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1012860108 Sczepanski et al. Downloaded by guest on September 24, 2021 data are consistent with significant involvement of histone H4 globular domain residues in the accelerated cleavage of AP sites. Examination of the nucleosome core particle crystal structure re- veals that lysine residues 20 and 31 of histone H4 are in proximity to AP89 and AP207, respectively, making these residues attractive candidates for Schiff base formation. Other proteins may also participate in this reaction, which is evident by an increase in H3 cross-linking at AP207 upon removal of the H4 tail. AP207 is in closer proximity to the H3 tail than is AP89. Additional experiments involving site-specific mutagenesis of H4 and liquid chro- matography/mass spectrometry of digested DPCs are required to determine exactly which lysine residues are responsible for these reactions. Overall, the data are consistent with a mechanism (Scheme 2) in which strand scission within the core particles is accelerated via Schiff base formation. Cleavage via Schiff base formation is consistent with model studies on AP lesions and randomly generated oxidized abasic sites in chromatin, which suggested that the core histone proteins significantly destabilize such lesions (26–28, 37, 38). Presumably, Schiff base formation is reversible. However, the data for AP disappearance fitted well to a simple first-order pro- cess. This suggests that the rate of Schiff base hydrolysis, reverting to an AP site, is probably slow compared to its formation and subsequent reaction. The presence of large amounts of cleaved DNA in isolated DPCs formed throughout the reaction of 1a (AP89) indicated that strand scission was rapid upon Schiff base Scheme 3. Postulated templating effect for the reaction of a bistranded AP formation. Finally, the persistence of DPCs after DNA strand scis- lesion with a single histone protein. sion is also consistent with relatively slow Schiff base hydrolysis. BIOCHEMISTRY Although a number of base excision repair proteins and DNA The above data indicate that in addition to longer lifetimes, polymerases exhibit lyase activities toward AP sites (39–42), it clustered lesions may be even more deleterious in cells due to is less common for proteins that are not enzymes to react with increased reactivity. Although AP sites are destabilized in nucleo- ∼4 DNA. In a recent rare example of the latter, Ku excised AP sites somal DNA, reaction at AP207 is accelerated -fold when it is from DNA via a Schiff base intermediate (43). Although AP clea- part of a bistranded lesion compared to when it is the sole lesion vage in a nucleosome core particle also proceeds via Schiff base in a core particle. The enhanced reactivity can be attributed to a formation, we would be remiss not to acknowledge that minor templating effect between two or more lysine residues of histone CHEMISTRY contributions to strand scission by other mechanisms that do not H4, where formation of the first Schiff base increases the effective involve direct attack by a lysine residue cannot be ruled out. molarity of other lysine side chains with respect to the AP site on The lifetime of an isolated AP site in a nucleosome is vastly the opposing strand (Scheme 3). This proposal is supported by experiments in which we measured histone H4 cross-linking shortened compared to that in free DNA, but rates of DPC for- 3a 3c mation and strand scission are modest compared to anticipated with AP89 ( ) and AP207 ( ) individually (when incubated in the presence of NaBH3CN). We determined that the sum total repair rates in cells. Following oxidative treatment, AP site levels 6 of the individual cross-links was greater than the total amount are restored to background levels (∼5 AP sites∕10 nucleotides of cross-linked duplex (Fig. 6B). This indicates that both AP sites or ∼30;000 AP sites∕cell) within 1 h in mammalian cells (11). We were cross-linked to the same protein molecule in a significant find that as many as 10% of the AP sites formed at SHL 1.5 re- fraction of nucleosome core particles. The reduction in DNA– sulted in DPCs with the nucleosome in the first hour. When one protein cross-linking and double strand break formation in the considers the above noted steady-state levels of AP sites per cell, presence of tailless histone H4 illustrate the importance of the it is likely that thousands of DNA-histone cross-links are formed lysine-rich H4 tail on these processes. per cell each day. Even greater production is expected in cells Accelerated double strand break formation from clustered exposed to exogenous forms of oxidative stress. Moreover, abasic lesions in nucleosomes is biologically important in its own clustered DNA lesions, which are a hallmark of ionizing radia- right, due to their highly deleterious nature. However, the lysines tion-induced damage, are repaired significantly more slowly than of the H4 tail that play such a large role in DNA cleavage at AP – isolated lesions (9, 35, 44 46). Consequently, clustered lesions sites near SHL 1.5 are often modified in cells (49, 50). Lysine containing AP sites are longer lived and may have significant bio- acetylation and permethylation will reduce the ability of these logical consequences including greater yields of highly deleter- side chains to induce strand scission of DNA containing AP sites. ious double strand breaks in addition to the DPCs noted above The strong involvement of H4 tails may indicate a connection (10, 47, 48). between double strand break formation and chromatin remodeling. It is well known that histone proteins undergo modification when DNA is damaged and this facilitates repair protein access to the damaged biopolymer (51, 52). Our observations raise the question whether it is possible that cells also modify H4 tails in response to DNA damage in order to protect against single and double strand breaks. Materials and Methods Nucleosome core particles were used directly in these experiments without Scheme 2. Reaction mechanism for the formation of a DPC and strand adjustments in the concentration following reconstitution (10 mM Hepes, break from AP in a nucleosome core particle. 60 mM NaCl, 1 mM EDTA, and 0.1 mM PMSF, pH 7.5). Nucleosome core

Sczepanski et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 24, 2021 particles containing 4 were photolyzed (350 nm) for 10 min at room tempera- lyzed by 10% native SDS PAGE (acrylamide∕bisacrylamide; 29∶1) as previously ture and immediately incubated at 37 °C for the duration of the time course described. SDS PAGE analysis of aliquots from 3a–3d was carried out using a experiment. For samples incubated in the presence of NaBH3CN (10 mM), the gel comprised of a 15% resolving layer (acrylamide∕bisacrylamide; 29∶1) and reducing reagent was added prior to photolysis. Aliquots were removed at a 3% stacking layer (acrylamide∕bisacrylamide; 29∶1). appropriate times and divided into two portions. One portion was treated with proteinase K (2 μg∕1 μL sample) for 5 min at room temperature and ACKNOWLEDGMENTS. We are grateful for support from the National Institute quenched by the addition of NaBH4 (0.1 M final). These samples were analyzed by 8% denaturing PAGE (acrylamide∕bisacrylamide; 19∶1, 45% urea). of General Medical Sciences (GM-063028 to M.M.G. and GM-084129 to G.D.B.). The second portion was treated directly with NaBH4 (0.1 M final) and ana-

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