Rotational Dynamics of DNA on the Nucleosome Surface Markedly Impact Accessibility to a DNA Repair Enzyme

Rotational dynamics of DNA on the nucleosome surface markedly impact accessibility to a DNA repair enzyme

John M. Hinz, Yesenia Rodriguez, and Michael J. Smerdon1

Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved January 26, 2010 (received for review December 16, 2009) Histones play a crucial role in the organization of DNA in the Although the density of chromatin is an essential factor for its nucleus, but their presence can prevent interactions with DNA role in organizing DNA, its presence can be problematic for binding proteins responsible for repair of DNA damage. Uracil is an normal DNA metabolic processes, including DNA repair and abundant mutagenic lesion recognized by uracil DNA glycosylase transcription (8, 10, 12–15). DNA associated with nucleosomes is (UDG) in the first step of base excision repair (BER). In nucleosome constrained, and the general helical mobility necessary for access core particles (NCPs), we find substantial differences in UDG- to binding factors is impeded (12). In addition, the DNA in directed cleavage at uracils rotationally positioned toward (U-In) direct contact with histones is occluded from interaction with or away from (U-Out) the histone core, or midway between these other proteins, as intervals of the helix along the histone core orientations (U-Mid). Whereas U-Out NCPs show a cleavage rate surface are buried away from the solvent and thus virtually just below that of naked DNA, U-In and U-Mid NCPs have markedly inaccessible (13, 16). slower rates of cleavage. Crosslinking of U-In DNA to histones in There are two primary “active” mechanisms used by the cell, NCPs yields a greater reduction in cleavage rate but, surprisingly, acting independently or in combination, to overcome the yields a higher rate of cleavage in U-Out NCPs compared with nucleosome-associated barriers to DNA metabolism. Chromatin uncrosslinked NCPs. Moreover, the next enzyme in BER, APE1, structure can be rearranged by ATP-dependant chromatin stimulates the activity of human UDG in U-Out NCPs, suggesting remodeling complexes which can remove, replace, and reposi- GENETICS these enzymes interact on the surface of histones in orientations tion nucleosomes along the DNA (17). In addition, histones are accessible to UDG. These data indicate that the activity of UDG targeted for a wide range of posttranslational modifications, likely requires “trapping” transiently exposed states arising from including methylation and acetylation, which can alter DNA– the rotational dynamics of DNA on histones. histone interactions and/or “mark” NCPs, for recognition, to aid additional activities (10). chromatin | DNA damage | glycosylase | histones | APE1 endonuclease Importantly, intrinsic nucleosome dynamics may serve a “pas- sive” role for contributing to exposure of otherwise inaccessible NA repair is essential for cell survival and prevention of sites on DNA. For example, nucleosomes exhibit spontaneous Dmutagenesis (1). Base excision repair (BER) is a key member transient (partial) unwrapping in vitro (18–20). Thus, even with- of the cellular DNA repair mechanisms and is responsible for out active nucleosomal displacement and modification, DNA detection of a wide range of prevalent DNA lesions, including repair proteins may use NCP dynamics for surveillance of DNA to alkylated nucleobases and uracil (2, 3). The first step of BER is detect damaged bases within nucleosomes. This is particularly recognition of the modified base by a glycosylase, which cleaves relevant for the initiation of BER, in which a glycosylase could the N-glycosidic bond and excises the base from its deoxyribose have difficulty accessing certain bases along the helix because of sugar, leaving an abasic (AP) site. Because the AP site repair their orientation relative to the histones surface. intermediate is a liability to the cell, some glycosylases are The effect of lesion orientation and the impact of spontaneous retained at the site with high affinity, released only when the next molecular motions promoting BER have been examined in factor in BER, the AP endonuclease (APE1), is available (4). previous studies attempting to understand glycosylase accessi- APE1 specifically recognizes AP sites in the DNA and incises the bility to their substrates in chromatin. Two studies assessing phosphodiester bond 5′ of the abasic residue. This single strand uracil DNA glycosylase (UDG) activity on uracil-containing break is typically the substrate for DNA polymerase β (Pol β), DNA assembled in NCPs showed reduced rates of UDG activity, which exerts DNA-deoxyribophosphodiesterase activity to cleave ranging from 3- to 10-fold (21, 22). However, these studies differ off the abasic sugar and uses the intact strand as template to in their conclusions regarding the orientation effects of uracil synthesize the missing base (5). Repair of the lesion is complete relative to the histones, as only the latter study showed a dif- after ligation of the newly synthesized strand, usually by DNA ference in UDG activity between “outward-” and “inward-” ligase III (6). Although these enzymatic steps can be accom- oriented uracil residues (22). plished by purified enzymes on naked DNA in vitro, there are The bifunctional glycosylase hNTH1 also shows orientation- accessory factors in cells, including XRCC1, that ensure efficiency driven DNA cleavage activity in NCPs assembled with thymine and coordination of the repair process (6, 7). Importantly, BER in glycol–containing DNA (23). Increased cleavage was measured the cell must take place on DNA in the context of chromatin. at lesions facing away from the histone surface versus those Chromatin is a dynamic structure, consisting of DNA and numerous proteins that fold and pack the genetic material at many levels to maintain organization and to regulate gene Author contributions: J.M.H. and M.J.S. designed research; J.M.H. and Y.R. performed re- expression (8–10). The primary level of chromatin packaging is search; J.M.H., Y.R., and M.J.S. analyzed data; and J.M.H., Y.R., and M.J.S. wrote the paper. the nucleosome core particle (NCP), consisting of 147 bp of The authors declare no conflict of interest. DNA wrapped around an octamer of the four core histones This article is a PNAS Direct Submission. H2A, H2B, H3, and H4 (11). These nucleosome cores are Freely available online through the PNAS open access option. located in close proximity along the chromosomes, separated by 1To whom correspondence should be addressed. E-mail: [email protected]. “ ” ∼ – short, variable lengths of linker DNA ( 20 90 bp), which are This article contains supporting information online at www.pnas.org/cgi/content/full/ bound by either of the linker histones H1 or H5. 0914443107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914443107 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 facing in, and differences between orientations were less pro- nounced at positions closer to the end of the DNA (23). Among these glycosylase studies it is evident that increasing the glycosylase concentration reduces the histone-associated inhibition of activity (21–23). Together, the hNTH1 and UDG data support the idea that glycosylases are sterically occluded from access to their respective substrates by the presence of histones. In addition, they suggest that glycosylases may gain accessibility to Fig. 1. Uracil orientation in NCP substrates. Positioning of uracil residues in lesions based on molecular motion within the nucleosome core, DNA on the surface of the histone octamer [figure created using PyMOL; as measured previously using restriction enzyme accessibility to structure taken from Davey et al. (54), Protein Data Bank Code 1KX5]. monitor the unwrapping of DNA near the NCP edges (18, 19). Curiously, measurements of the time in which regions of the rotation of a DNA strand (Fig. S2). To determine the orientation DNA along the nucleosome are unwrapped vary greatly, from of uracil in the reconstituted NCPs, the three different constructs the ends to the center of the histone octamer (dyad axis), by a were digested with UDG and APE1, which cleaves the DNA factor of ∼10,000-fold (20). With such a great difference in backbone specifically at the uracil residues, and compared the exposure based on unwrapping, it is surprising that the glycosylase studies on NPCs show only modest differences in activity location of the digestion products with the NCP footprint (Fig. between inward and outward orientations, especially near the S2). Peak analysis of the scan provides a clear visualization of the dyad center of the DNA. comparison between the NCP pattern and the location of the In this study, we determined the impact of histones on the uracil residues within the digested DNA substrates (Fig. 2). The recognition step of BER by measuring UDG-directed DNA data indicate that the relative orientations of the uracils in the cleavage (with or without APE1) at uracil residues carefully po- U-In, U-Mid, and U-Out NCPs accurately represent their re- sitioned near the dyad axis of NPCs in three distinct orientations. spective orientations on the histone surface. Using a strong NCP positioning sequence and hydroxyl radical footprinting, we verified that uracils in the U-In, U-Out, and U- Unbiased UDG/APE1 Activity at Uracils in Different Sequence Contexts. It has been reported that the rate of glycosylase activ- Mid NCPs were oriented toward the histone core, toward the fi solvent, and midway between these orientations, respectively. To ity associated with repair of a modi ed nucleobase can be greatly determine the impact of DNA rotational dynamics in the NCPs on affected by DNA sequence context (28, 29). Therefore, we UDG activity, we used formaldehyde crosslinking to suppress treated the naked uracil-containing DNAs with limiting amounts DNA movement on the histone octamer surface. Finally, we (0.2 nM) of UDG and APE1 to determine their relative rates of assessed the potential role of AP endonuclease in enhancing gly- cleavage. After treatment over a course of 60 min, it was clear cosylase activity in the context of histones by comparison of human that the uracils in the U-In and U-Out DNA were digested at UDG (hUNG2) activity at each uracil orientation in the presence very similar rates, whereas the U-Mid DNA construct showed a or absence of APE1. slightly lower rate of initial cleavage (Fig. S3). Importantly, there appears to be little difference in the relative rates of cleavage Results among the DNAs, eliminating the possibility that the sequence Reconstitution and Verification of Nucleosome Core Particles with context of the uracil residues accounts for any measured differ- Uracil Residues in Specific Rotational Orientations. To create stable ences in the enzymatic cleavage of these DNAs associated mononucleosome substrates with particular orientations of uracil with NCPs. bases relative to the histone surface, we used synthesized 150 base oligonucleotides containing the 15 base canonical glucocorticoid Extreme Range of UDG/APE1 Activity Among Different Uracil receptor response element (GRE) sequence flanked by the well- Orientations in NCPs. To determine the effect of different uracil characterized nucleosome positioning TG motif sequences (TG- orientations on the activity of UDG/APE1, we examined the GRE-TG) (16, 24, 25). Three different cytosine bases within the time courses of cleavage of the three DNA substrates in NCPs. GRE were replaced with uracil, labeled U-In, U-Mid, and U-Out (Materials and Methods), in reference to their predicted orientations relative to the histone octamer determined by the TG positioning motifs. Cytosine residues were chosen because of the biological relevance of replacement by uracil from naturally occurring spontaneous cytosine deamination, leading to G:U mispairs in DNA (26). We also created a strand that encoded the native GRE sequence without uracil (UND). The oligos with uracil were labeled with 32P at the 5′ end and annealed with the complementary strand to create the double strand DNA substrates used in this study. The structure of the histone octamer at the dyad axis and the predicted orientations of the uracil residues after pairing with their complementary strand are shown in Fig. 1. Uracil-containing NCPs were reconstituted from purified chicken erythrocyte mononucleosomes using salt dialysis, as Fig. 2. Hydroxyl radical footprint of reconstituted nucleosome core particles described (25). As expected, the labeled TG-GRE-TG DNA and relative uracil positions in the DNA substrate. Figure shows scans of a assembled efficiently with the histone octamers, independent of sequencing gel (Fig. S2) with the hydroxyl radical footprint of the undamaged the presence or location of uracil in the sequence (Fig. S1). To DNA substrate associated with the histone octamer and the relative uracil verify the rotational orientation of the uracil residues, we per- positions of the U-In, U-Mid, and U-Out DNA constructs, after treatment with UDG and APE1. A labeled 10-bp DNA marker is also scanned for relative formed hydroxyl radical footprinting on reconstituted NCPs (27). positioning, and the inverted triangle marks the dyad center of the NCP. Note When this method is performed on NCPs, the reaction products that the actual position of the uracil residues of each substrate, in comparison separated on a sequencing gel reveal the regions of the DNA with the NCP footprint, will be one nucleotide longer (shifted one peak to the backbone that are exposed to solvent from those associated with left), as the APE1 endonuclease cleaves on the 5′ side of the lesion, between the histones, yielding a periodic pattern reflecting the helical the uracil residue and the 5′ radiolabeled end.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0914443107 Hinz et al. Downloaded by guest on September 28, 2021 For each of the NCP species, two UDG concentrations were is roughly equivalent to that seen for U-Mid NCPs at 100-fold used (with APE1 in equimolar concentrations to UDG), and UDG concentration (20 nM) (Fig. 3; compare Middle and cleavage of the DNA was assessed over the course of 1 h on Lower). These data highlight the vast differences in the impact of denaturing polyacrylamide gels (Fig. S4). Quantification and histone binding on activity of the glycosylase due to the ori- plotting of the percent cleavage as a function of time revealed entation of the lesion. that the difference in UDG/APE1 activity was so large between constructs that they could not be compared directly at a common Restricting Movement of DNA in NCPs Changes UDG Activity. The UDG concentration (Fig. 3). For example, at 2 nM UDG, there DNA in NCPs is dynamic on the surface of histone octamers is measurable cleavage activity among the NCPs and their leading to transient, variable states of unwrapping that may per- respective naked DNAs, but cleavage of the U-Out naked DNA petuate along the entire length of a DNA strand (18–20), and it is and NCP is virtually saturated (Fig. 3). By reducing the UDG/ likely that some movement of the DNA is present throughout the APE1 concentrations 10-fold, a difference between the U-Out nucleosome core (30). To explore the impact of DNA rotational naked DNA and the U-Out NCP is seen (Fig. 3, Lower). Thus, dynamics on the activity of UDG/APE1 cleavage in our NCPs, we the outward oriented uracil shows a histone-associated decrease restricted movement of the DNA on the surface of the histone in UDG activity when the amount of UDG is reduced to a octamers using formaldehyde crosslinking (31, 32). NCPs were reaction-limiting concentration. treated with 1% formaldehyde for 1 h, followed by subsequent The U-In and U-Mid NCPs showed extensive reduction in the crosslink quenching with glycerol and dialysis. The formation of formation of cleavage products relative to their naked DNA covalent bonds between the DNA and histones was verified on controls, with U-In showing the greatest reduction (Fig. 3, denaturing gels, in which no evidence of free DNA was found in Upper). Some of this cleavage inhibition was overcome by samples after the crosslinking procedure (Fig. S5). increasing the UDG/APE1 concentration by 10-fold (20 nM), Formaldehyde crosslinking did not affect the activity of UDG/ but still did not show the formation of cleavage products close to APE1 on naked DNA but, surprisingly, did lead to increased that of the naked DNAs at 2 nM (Fig. 3, Upper and Middle, cleavage of the U-Out NCP (Fig. 4, Upper), bringing the rate of respectively). Importantly, although APE1 is used in the reaction cleavage closer to that of naked DNA. This indicates that in the to create the strand breaks necessary for formation of the orientation associated with U-Out, UDG/APE1 activity is opti- cleavage products, UDG is the rate limiting factor in these mal, and any movement of the DNA on the histone surface during measurements, as we found that reducing the APE1 concen- the course of the reaction presents less favorable orientations. GENETICS tration in the reaction to as low as 1% of the UDG concentration Conversely, crosslinking caused a dramatic decrease in the did not effect the rates of substrate cleavage. To get a sense of cleavage rate of the U-In NCPs, virtually eliminating digestion the relative differences in UDG activity among the NCPs, for- after the first 5 min (Fig. 4, Lower). The early appearance of U-In mation of cleavage products in the U-Out NCPs at 0.2 nM UDG cleavage products may be due to “locking” of some of the uracils in the histone-crosslinked DNA into more accessible orientations in a small fraction of the NCPs. Because formaldehyde treatment occurs over 1 h, the DNA dynamics in NCPs allow some crosslinks to “lock in” less frequent topological states. This may be affecting U-Out NCPs as well, in which some of these NCPs may be crosslinked in less favorable conformations (flattening the cleavage curve in the crosslinked U-Out sample, as in Fig. 4,

Fig. 3. Assessment of UDG and APE1 activity on NCP substrates with different uracil orientations. Plots of relative rates of cleavage of each DNA/ NCP substrate (U-In, U-Mid, and U-Out in Upper, Middle, and Lower, respectively) over a 1-h time course. Dashed lines represent naked DNA of Fig. 4. UDG and APE1 cleavage of formaldehyde crosslinked NCPs. (Upper) each construct; solid lines represent NCPs. UDG concentrations are listed on Time course after Incubation with 0.2 nM UDG of Naked DNA (squares) and the Right Insets. For each construct, the data for naked DNA and NCPs U-Out NCPs (triangles) with and without 1 h preincubation with form- incubated with 2 nM UDG is denoted by open boxes and open diamonds, aldehyde (open symbols/dashed lines and closed symbols/solid lines, respectively. Data for NCPs incubated with 20 nM UDG (For U-In and U-Mid respectively). (Lower) Time course after incubation with 20 nM UDG of U-In only) are denoted by open triangles; data for naked DNA and NCPs incu- (circles) and U-Mid NCPs (diamonds) with and without 1 h preincubation bated with 0.2 nM UDG (U-Out only) are denoted by open and closed circles, with formaldehyde (open symbols/dashed lines and closed symbols/solid respectively. Each data point represents the mean ± 1 SD of at least three lines, respectively). Each data point represents the mean ± 1 SD of at least independent experiments. three independent experiments.

Hinz et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Upper). Finally, the U-Mid NCPs did not appear to be sig- uracil residues in NCPs, and that the absence of APE1 does not nificantly affected by formaldehyde treatment (Fig. 4, Lower). abrogate the effect. This may reflect the movement of uracil into both favorable and To examine the possibility that the AP endonuclease may unfavorable transient states with roughly equal but opposite promote glycosylase activity in the context of histones, we impacts on UDG accessibility. Thus, preventing U-Mid from repeated hUDG activity experiments in the presence of equi- vacillating into these different orientations would have a minimal molar concentrations of APE1. APE1-enhancement of hUDG effect on the rate of cleavage. activity is clearly measurable on naked DNA substrates, enhancing the rate of cleavage by ∼2- to 3-fold (Fig. 6, dashed APE1 Stimulates hUDG Activity on NCPs. Some glycosylases have lines). It also appears that hUDG activity is enhanced by the very high affinity for AP sites (their reaction product), and their presence of APE1 on U-Out NCPs, although not to the same rate of activity is reduced over time by the removal of free gly- extent as naked DNA (∼1.3- to 1.5-fold). However, unlike the cosylase enzymes from the reaction, a process known as “product naked DNA and U-Out NCPs, the enhancement of hUDG inhibition” (33, 34). APE1 has been shown to enhance efficiency activity in the presence of equimolar concentrations of APE1 was of these glycosylases by releasing the enzyme from its product or not detected in the U-In and U-Mid NCPs (Fig. S6). Thus, the preventing rebinding to the AP site, and thereby recycling the association of DNA with histones does not eliminate APE1- enzyme for binding to other lesions. APE1 has also been shown dependent enhancement of hUDG activity in the solvent-exposed to enhance the efficiency of human UDG (hUDG, commonly uracil orientation. known as hUNG2) (35), presumably by the same mechanism. Discussion Although it is not known whether APE1 stimulation of hUDG occurs in the context of NCPs, the presence (or absence) of Glycosylase recognition of a damaged substrate initiates the BER process by direct lesion binding. Bulky chemical adducts APE1 in previous assessments of UDG activity on NCPs has and UV light–induced pyrimidine dimers, repaired by nucleotide been suggested to be a factor in discrepancies associated with the excision repair (NER), lead to distortions in the topology of the impact of uracil orientation (36). Whereas UDG activity DNA helix. Some of these lesions can alter nucleosome structure appeared to be unaffected by uracil orientation when measuring (25, 37) and many disrupt transcription elongation complexes (8, uracil base excision (done in the absence of APE1) (21), meas- 38–40). However, most chemical modifications recognized by uring cleavage of the DNA as an indicator of UDG activity (with glycosylases cause comparatively small perturbations in DNA, APE1) has shown orientation dependence in this study and with little evidence for preferential nucleosome perturbations or, elsewhere (22). We wished to determine whether APE1 stim- in most cases, blockage of transcription (41–43). Thus, in the ulates hUDG activity in the context of NCPs, in addition to cell, there appears to be a heavy reliance on intrinsic accessibility determining whether the effect of histones on UDG activity is to damaged chromatin-associated DNA bases by glycosylases. independent of APE1. In this study, we found extreme differences in the rates of UDG fi fi We rst veri ed that hUDG shows the broad range of activity activity dependent on the orientation of the uracil relative to the among the uracil orientations seen in the recombinant E.coli histone surface near the dyad center of NCPs, pointing to a clear UDG used in the previous experiments. We measured the rel- effect of lesion proximity to histones. The effect of orientation in ative rates of cleavage among the different NCP and naked DNA our study was substantially greater than that measured in previous substrates in the absence of APE1 to prevent the potential for an studies on UDG and hNTH1 (21–23). However, after submission APE1 effect on the activity of hUDG. To reveal hUDG activity, of this manuscript, another report appeared showing a reduction the DNA was purified after incubations with the catalytic domain of ∼3,000- to ∼10,000-fold in UDG activity at uracils oriented of hUDG and treated with APE1 to allow complete cleavage at toward histones in NCPs (44), in close agreement with our results. all abasic sites in the DNA. As with E. coli UDG, we found a Variation in DNA sequence may account for some of the dif- large difference in formation of cleavage products between the ferences among the previous studies, including the use of Lyte- U-In and U-Out NCPs, with U-Mid NCPs closely mimicking the chinus variegatus 5S rDNA as the NCP positioning sequence (21, U-In orientation (Fig. 5). Also, like the E. coli enzyme, a 10-fold 23). This sequence has reduced histone binding energy and increase in hUDG concentration (20 nM) was necessary for the multiple translational settings compared to the sequence used in U-In and U-Mid NCPs for reliable quantification over the time this study (45). To improve homogeneity in our NCP prepara- course (at 20 nM UDG, both naked DNA and U-Out NCP tions, we used the strongly binding TG nucleosome positioning samples are completely cleaved within 15 min). Thus, it is clear sequence. The minimal impact of uracil orientation in a previous that human UDG is also greatly affected by the orientation of study that also used the TG positioning motifs (22) is explained in a subsequent study from our laboratory, in which the actual ori-

Fig. 5. Relative rates of hUDG and APE1-directed cleavage DNA and NCP constructs. Naked DNA (open triangles with dashed line) and NCP substrates Fig. 6. Influence of APE1 on hUDG activity in naked DNA and U-Out NCPs. U-In (small filled squares), U-mid (filled triangles), and U-Out (large filled Naked DNA (triangles and dashed lines) or U-Out NCPs (circles and solid squares) are represented. The hUDG concentration for the naked and U-Out lines) incubated with hUDG in the presence (filled symbols) or absence (open NCPs is 2 nM, and for the U-In and U-Mid NCPs is 20 nM. Each data point symbols) of APE1. Each data point represents the mean ± 1 SD of at least represents the mean ± 1 SD of at least three independent experiments. three independent experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0914443107 Hinz et al. Downloaded by guest on September 28, 2021 entations of the uracil positions used were assessed by hydroxyl uracil-containing NCP substrates. Thus, whereas turnover of the radical footprinting (46). We found that the two orientations used limited amount of hUDG is important for maintaining high rates by Beard et al. (22) may not have represented truly “in” and “out” of cleavage in the naked DNA and U-Out NCPs (where the orientations but, rather, shifted one or two bases from the vertical enzyme to substrate ratio is close to unity), any APE1-associated axis (46). Considering the wide range of orientations possible on effect on hUDG recycling during the U-In and U-Mid NCP the surface of histones for different sequences, assessment of the reactions would provide a negligible increase in the available pool NCPs via hydroxyl radical footprinting was critical for our choice of hUDG in the reaction. This punctuates the conclusion that, in of base orientation in this study. the U-In and U-Mid NCPs, it is the inability of the glycosylase to The crosslinking experiments proved to be particularly access the lesion that is responsible for the reduction of hUDG revealing of the importance of DNA dynamics on histones in activity between the unfavorable uracil orientations. exposing unfavorable base orientations to the solvent. As There are undoubtedly many modes of motion among the expected, the crosslinked U-In NCP showed very little UDG protein and DNA molecules that make up chromatin. In the activity, at least after the ﬁrst 5 min, highlighting the necessity for more open nucleosome-loaded regions, the intrinsic, sponta- DNA movement on the histone surface for transient exposure of neous dynamics of nucleosomes are likely contributing to the unfavorable helical orientations. control of DNA binding factors and enzyme access to DNA, Surprisingly, the U-Out NCPs showed an increase in the rate of including proteins responsible for recognizing DNA lesions. Our DNA cleavage after crosslinking, suggesting that crosslinking results suggest that at least some regions of DNA in chromatin restricts movement of the DNA that restrains some bases to are likely afforded little access to DNA-binding proteins, and favorable positions. Indeed, results with the U-Mid NCP, which that rotational motion of the DNA may be the primary mecha- showed little difference between the crosslinked and non- nism by which glycosylases can access these buried sites. Fur- crosslinked states, point to a similar conclusion. Our data showing thermore, the marked difference in rates of glycosylase activity increased DNA cleavage in U-Mid NCPs with a higher UDG among the uracil orientations indicate a potential persistence of concentration suggest transient states of increased glycosylase lesions facing toward the histones and thereby inﬂuencing accessibility. However, because immobilization of the DNA did mutagenesis. Indeed, recent studies have found the highest not affect the measured UDG activity on U-Mid NCPs, uracil occurrence of single nucleotide polymorphisms (SNPs) among residues in the U-Mid orientation in noncrosslinked NCPs must human CpG sequences in regions of closed chromatin (49), and

be vacillating between both more accessible and less accessible a 146-nucleotide periodicity of SNPs in transcription start sites GENETICS orientations. Thus, our crosslinking results suggest that, at least among humans (50). These results may reflect the conserved near the dyad center of NCP DNA, access to uracils by UDG is placement of nucleosomes in these regions, and point to the driven more by rotational dynamics of DNA on the histone sur- direct impact of histones on mutation rate. face than by transient unwrapping. The decrease in glycosylase activity that we found for the Materials and Methods outward oriented lesion, relative to naked DNA, despite the Detailed methods are provided in SI Text. otherwise “optimal” positioning, was also seen in the previous glycosylase studies using NCPs (21–23). One explanation is that Oligonucleotide Synthesis, 32P Labeling, and NCP Reconstitution. Double- this may be due to the interference by the histones of one- strand DNA substrates for study contained a central 15 bp GRE sequence dimensional glycosylase “scanning” along the DNA, a mecha- flanked by the TG nucleosomal positioning sequences (13, 24, 25), and were nism proposed for detection of damaged substrates (35, 47, 48). created by annealing of 150 base oligonucleotides (synthesized by solid Our crosslinking results, showing increased UDG activity in the phase synthesis by Midland Certified Reagent Company). Three of the oligos immobilized U-Out NCPs, approaching the rates measured for (U-IN, U-MID, and U-OUT) each contained uracils at a different position naked DNA, suggest that at least some of the histone-associated within the GRE; one oligo (UND) had the same GRE sequence with no uracils; glycosylase inhibition is due to transient rotation of uracil into and one oligo (PARTNER) served as the partner strand for annealing with the rest. Sequences of the GRE at the center of each of the four oligos are as less accessible orientations. However, we cannot eliminate the follows: GRE: TGTACAGCATGTTCT; U-In: TGTACAGCATGTTUT; U-Mid: possibility that a decrease in glycosylase scanning may account TGTAUAGCATGTTCT; U-Out: TGTACAGUATGTTCT. Before annealing with for some of the reduced UDG activity on NCPs, as the rate of the partner strand, oligos were radiolabeled with 32Patthe5′ ends with T4 cleavage in the U-Out NCPs did not equal that measured with polynucleotide kinase (Invitrogen) and γ-[32P]ATP (PerkinElmer) for 30 min naked DNA, even after crosslinking. at 37° C. Mononucleosomes were prepared by histone octamer transfer, The high affinity of glycosylases for AP sites is thought to be an combining the radiolabeled 150-bp DNA substrates with chicken erythrocyte important factor in protecting these sites, and potentially core particles prepared from chicken erythrocytes (51) at high ionic strength, recruiting the next enzyme in the BER pathway (APE1). This and subsequent incremental diaylsis as described previously (25, 46). process of presenting the product of each reaction as a viable substrate for the next repair step is referred to as “passing the UDG/hUDG and APE1 Digestion. Treatment of DNAs and NCPs with recombi- baton” (4). This accounts for the product inhibition associated nant E.coli UDG (Ung; New England Biolabs) or the catalytic domain of “ ” hUDG (hUNG2 with N-terminal deletion of 84-amino acids entailing single- with UDG activity, which is overcome by the hand-off of the strand DNA and RPA binding domains (52); kindly provided by Samuel abasic site to the AP endonuclease. We saw clear evidence of the Wilson (National Institute on Environmental Health Sciences) and human APE1 stimulation of hUDG activity in the naked DNA and APE1 (New England Biolabs) were initiated by adding UDG or hUDG and APE U-Out NCP, indicating that the association of DNA with histones to final concentrations of 0.2, 2, or 20 nM each, with APE1 concentrations does not greatly affect the ability of AP endonuclease to remove always equal to UDG concentrations unless otherwise specified. Incubations the glycosylase from the abasic repair intermediate, at least at an were at 37°C for 5, 15, 30, and 60 min. Reactions were terminated with AP site facing away from the histone core. Because we did not addition phenol:chloroform:isopropanol (PCI; 20:19:1) at equal volume to find an APE1-associated increase in cleavage of the U-In and the reaction, and DNA recovered by ethanol precipitation. For reactions with U-Mid NCPs (Fig. S5), it is tempting to speculate that, at hUDG in the absence of APE1, the reaction conditions were run as described unfavorable orientations, there is a histone-associated impair- above without APE1. After DNA isolation, a second digestion reaction was run on the naked DNA, with only APE1 at 2 nM for 30 min at 37°C to reveal ment of the endonuclease to recycle the glycosylase from its all of the abasic sites. All naked DNA samples had chicken erythrocyte core product, or that APE1 is prevented from accessing buried abasic particles added to a concentration of 300 pM to adjust for the excess core sites. However, the 20-nM concentration of hUDG in the U-In particles present in reconstituted nucleosome samples. Digested DNAs were and U-Mid NCP cleavage assays (necessary for quantifiable separated on 10% polyacrylamide (0.5% bisacrylamide) 7M Urea denaturing detection of cleavage) is in vast excess (10-fold) relative to the gels in 1× TBE buffer, exposed to PhosphorImager screens (Molecular

Hinz et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Dynamics), visualized on a STORM 840 PhosphorImager (Amersham), and were then treated with high NaCl (to 1 M) for 5 h at 65°C to remove images were analyzed with IMAGEQUANT software (Molecular Dynamics). crosslinks. DNA was subsequently isolated by PCI and ethanol precipitation and separated and analyzed as described above. Hydroxyl radical foot- Formaldehyde Crosslinking and Hydroxyl Radical Footprinting. To covalently printing of the NCPs was performed as described previously (53). For uracil crosslink DNA to associated histones, NCPs were treated with 1% form- location control samples, DNAs containing uracil were each treated with 20 aldehyde (from 37% stock) on ice for 1 h, after which crosslinking activity was nM UDG and 10 nM APE1 and incubated at 37°C for 60 min. quenched by addition of glycerol to a ﬁnal concentration of 1%. Form- aldehyde- and mock-treated samples were dialyzed in NCP reconstitution ACKNOWLEDGMENTS. The authors thank Dr. Samuel Wilson, National buffer (10 mM Tris, pH7.5, 1 mM EDTA, 50 mM NaCl) for 1 h at 4°C to remove Institute on Environmental Health Sciences (NIEHS), for providing hUDG glycerol and formaldehyde. UDG/APE1 digestions were performed as and Dr. Raymond Reeves, Washington State University, for providing chicken described above, but reactions were terminated with addition of EDTA (to erythrocyte NCPs. This study was supported by National Institutes of Health 40 mM) and boiled for 1 min to eradicate repair enzyme activity. Samples (NIH) Grants ES004106 and ES002614 from the NIEHS.

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