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A Plug-and-Play Approach for Preparing Chromatin Containing Site-Specific DNA Modifications: The Influence of Chromatin Structure on Base Excision Repair Deb Ranjan Banerjee, Charles E. Deckard III, Meagan B Elinski, Meridith L. Buzbee, Wesley Wei Wang, James D. Batteas, and Jonathan T. Sczepanski J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04063 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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A Plug-and-Play Approach for Preparing Chromatin Containing SiteSpecific DNA Modifications: The Influence of Chromatin Structure on Base Excision Repair

Deb Ranjan Banerjee,‡,† Charles E. Deckard III,‡,†Meagan B. Elinski, ‡ Meridith L. Buzbee, ‡ Wesley Wei Wang‡, James D. Batteas, ‡ and Jonathan T. Sczepanski‡,* ‡

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United

States

*Corresponding Author [email protected]

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ABSTRACT The genomic DNA of eukaryotic cells exists in the form of chromatin, the structure of which controls the biochemical accessibility of the underlying DNA to effector proteins. In order to gain an in depth molecular understanding of how chromatin structure regulates DNA repair, detailed in vitro biochemical and biophysical studies are required. However, due to challenges associated with reconstituting nucleosome arrays containing site-specifically positioned DNA modifications, such studies have been limited to the use of mono- and dinucleosomes as model in vitro substrates, which are incapable of folding into native chromatin structures. To address this issue, we developed a straightforward and general approach for assembling chemically defined oligonucleosome arrays (i.e. designer chromatin) containing site-specifically modified DNA. Our method takes advantage of nicking endonucleases to excise short fragments of unmodified DNA, which are subsequently replaced with synthetic oligonucleotides containing the desired modification. Using this approach, we prepared several oligonucleosome substrates containing precisely positioned 2′-deoxyuridine (dU) residues and examined the efficiency of base excision repair (BER) within several distinct chromatin architectures. We show that, depending on the translational position of the lesion, the combined catalytic activities of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) can be either inhibited by as much as 20-fold or accelerated by more than 5-fold within compact chromatin (i.e. the 30 nm fiber) relative to naked DNA. Moreover, we demonstrate that digestion of dU by UDG/APE1 proceeds much more rapidly in mononucleosomes than in compacted nucleosome arrays, thereby providing the first direct evidence that inter-nucleosome interactions play an important role in

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regulating BER within higher-order chromatin structures. Overall, this work highlights the value of performing detailed biochemical studies on precisely modified chromatin substrates in vitro and provides a robust platform for investigating DNA modifications in chromatin biology.

INTRODUCTION The DNA of eukaryotic cells is packed into the hierarchal structure of chromatin. The basic unit of chromatin is the nucleosome, which is comprised of ~147 base pairs (bp) of DNA wrapped ~1.6-1.7 times around a protein core comprised of two copies of each histone H2A, H2B, H3, and H4. Individual nucleosomes are connected by stretches of linker DNA to form chromosome-sized oligonucleosome arrays that fold and condense into more compact higher-order structures (e.g. 30 nm fiber).1 Nucleosomes can restrict DNA-binding factors from accessing the underlying DNA (i.e. nucleosomal DNA).2,3 In addition, both nucleosomal DNA and the linker DNA become further occluded upon chromatin compaction.4,5 As a result, the structural state of chromatin influences a broad range of genomic processes, including transcription, recombination and DNA repair.6-8 Major efforts are now underway towards unraveling the molecular mechanisms underlying chromatin’s structure-function relationships. In recent years, chemically defined nucleosome arrays (often referred to as “designer” chromatin) have gained an increasingly important role in elucidating fundamental molecular mechanisms of chromatin regulation by enabling researchers to carry out quantitative measurements under precisely defined experimental conditions.9,10 In particular, the availability of multiple protein engineering strategies for introducing site-

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specific posttranslational modifications (PTMs) has enabled assembly of synthetic chromatin fibers harboring defined arrangements of modified histones.11-16 These chromatin substrates have been instrumental in revealing how specific histone PTMs alter DNA accessibility and local chromatin structure, in many cases providing molecular details that could not be obtained using simpler systems (i.e. mono- or dinucleosomes).1719

Designer chromatin approaches also provide an opportunity to investigate the functional relationship between chromatin structure and DNA modifications. For example, it is clear from work in many model systems that nucleosome organization and the degree of chromatin compaction play critical roles in regulating the repair of damaged DNA.20-23 Therefore, studies using well-defined chromatin complexes containing positioned DNA damage sites are expected to provide unprecedented molecular insight into the relationship between chromatin structure and DNA repair. However, in contrast to histone PTMs, methods for incorporating precisely modified DNA templates into designer chromatin are lacking, precluding such investigations. The key bottleneck is assembly of the large (>2,000 bp) double-stranded DNA (dsDNA) template containing a site-specific modification. This is challenging for at least two reasons: first, the majority of modified nucleotides are incompatible with enzymatic replication, including PCR amplification; and second, DNA polymerases are unable to install modified nucleoside triphosphates in a site-specific manner. As a result, long DNA templates harboring precisely modified sites are most often prepared using multi-step ligation-based strategies.24-26 For example, several shorter oligonucleotide fragments, one of which contains the modified nucleotide, can be prepared by solid-phase oligonucleotide

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synthesis and subsequently ligated together to form the desired DNA duplex. The efficiency of ligation, however, drops precipitously as the number of individual DNA fragments is increased, severely limiting the length of the DNA molecule that can be produced using this approach. While it is possible to further ligate short modified duplexes with additional DNA fragments in order to generate a larger DNA molecule, intermolecular ligation reactions typically require several purification steps, making the isolation of useable quantities of purified DNA difficult. Taken together, there remains a critical need to develop efficient strategies for generating site-specifically modified DNAs, especially those that are compatible with assembling oligonucleosome arrays. In the absence of a straightforward approach to prepare designer chromatin containing precisely positioned DNA modifications, in vitro studies aimed at uncovering the molecular mechanisms of DNA repair within chromatin have instead relied on modified mono- and dinucleosomes as model substrates.22,27 For example, numerous studies have characterized the activities of component enzymes involved in base excision repair (BER) on reconstituted mononucleosomes containing site-specifically damaged DNA.28-37 The BER pathway is responsible for removing the majority of endogenous DNA lesions in human cells38, and plays a critical role in active DNA demethylation.39 Although these studies provide insight into how BER occurs within the complex environment of chromatin, a single reconstituted nucleosome falls short of being an ideal chromatin substrate. For example, it is not possible to assess the impact of higher-order chromatin folding on BER enzyme activity using these model systems. Furthermore, the relationship between DNA repair and epigenetic modifications (e.g. histone PTMs), especially those shown to modulate chromatin structure, is difficult to address using

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mononucleosomes. Therefore, a deeper mechanistic understanding of DNA repair within chromatin will remain elusive until challenges associated with assembling sitespecifically damaged nucleosome arrays, which more accurately represent chromatin in vivo, are overcome. With the above considerations in mind, herein we describe a straightforward approach for preparing 12-mer oligonucleosome arrays (>2,000 bp) containing a single modified nucleotide incorporated at a specific site with nucleotide resolution. In contrast to cumbersome ligation-based strategies used previously, our approach enables rapid insertion of chemically modified oligonucleotides into nucleosome arrays via a simple strand exchange process. Using this “plug-and-play” approach, we assembled two different 12-mer oligonucleosome arrays each containing a single dU residue incorporated at a specific site. We then used these arrays to systematically examine the efficiency of BER initiation within various chromatin environments, including naked DNA, mononucleosomes, extended nucleosome arrays, and the 30 nm chromatin fiber. To the best of our knowledge, this is the first time the repair of a positioned DNA lesion has been studied within multiple hierarchical structures of chromatin. Beyond DNA repair, we imagine that the robust methods reported herein will find application in the study of other important DNA modifications, such as 5-methylcytosine and its oxidized derivatives, in chromatin.

RESULTS AND DISCUSSION Preparation of DNA templates containing site-specific modifications. The oligonucleosome arrays described in this report utilize a DNA template composed of 12

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copies of the 147-bp “601” nucleosome positioning sequence,40 with 30 bp of linker DNA separating consecutive nucleosome core particles (Figure 1a). Similar systems have been used to carry out detailed in vitro biophysical studies on chromatin dynamics,41,42 and to investigate the regulation of chromatin structure by histone PTMs.17-19 Importantly, this design exactly reproduces the architecture of the 12-mer nucleosome array whose structure was characterized by cryo-electron microscopy.43 Consequently, the molecular environment of individual nucleotides within the 30 nm chromatin fiber are known at single-nucleotide resolution.

Figure 1. Designer chromatin containing site-specific DNA modifications. (a) Schematic illustration of the 12×601 DNA templates. The exchangeable DNA strand fragments

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within nucleosome 5 (N5) (12-601-Nt, yellow box), N7 (12× ×601-Nt/SI, green box), or the adjacent linker DNA (12-601-Nb, orange box) are indicated. (b) The dU containing substrates prepared in this study. dU49 and dU88 (red dots) are positioned either 49 or 88 nucleotides from the N5 nucleosome dyad, respectively. (c) Cryo-electron microscopy structural model of the 30 nm chromatin fiber (12×601, EMD-2600) showing the locations of dU49 and dU88, as well as the corresponding Nt.BstNBI (yellow) and Nb.BbvCI (orange) excisable DNA regions. The nucleosome surface (PDB ID: 1ZBB) was fitted to the electron density map using Chimera.44 (d) Nucleosome-level view (N5) of dU49 and dU88 positioning (PDB ID: 1ZBB). See Supporting Information Text for DNA sequence information.

In order to facilitate construction of nucleosome arrays containing site-specifically modified DNA, we first focused on developing a straightforward approach for inserting modified nucleotides into the requisite 12×601 DNA templates. In particular, we sought to avoid the inefficient joining together of multiple DNA fragments using intermolecular ligation. Instead, we chose to employ a strategy whereby sequence-specific nicking endonucleases (or “nickases”) are used to nick the DNA at two proximal sites,45,46 resulting in formation of a short gap upon melting. This gap is then filled with a DNA oligonucleotide carrying the desired modification and the nicks subsequently resealed to generate an intact DNA template (Figure 2a). Because the sealing of DNA nicks by T4 DNA ligase is extremely efficient, we anticipated that this approach would eliminate some, if not all, purification steps. Hence the overall yield of modified DNA would be

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dramatically increased relative to previously employed ligation-based methods. Another advantage of this strategy is that it only requires synthesis of a single, short oligonucleotide fragment, potentially broadening the scope of designer chromatin by enabling incorporation of synthetically challenging DNA modifications. To demonstrate the feasibility of this approach, we prepared three different 12×601 templates containing excisable DNA fragment(s) within either the nucleosome-bound region of the 601 DNA (12-601-Nt and 12-601-Nt/SI) or the adjacent linker DNA not associated with the nucleosome core (12-601-Nb) (Figure 1a and Figures S1–S5). Because the accessibility of DNA within chromatin depends largely on nucleosome positioning,47 we anticipated that incorporating DNA modifications within these structurally distinct regions would facilitate future studies aimed at uncovering the structure-activity relationships of chromatin associated proteins. Moreover, substrate 12601-Nt/SI offers the possibility to incorporate two different modified oligonucleotides into the same oligonucleosome array. In order to preserve the structure of the modified 601 nucleosomes within 12-601-Nt and 12-601-Nt/SI (nucleosomes N5 and/or N7; Figure 1a), the proximal nickase recognition sites were positioned such that a minimal number of mutations were introduced into the 601 DNA sequence (Figure S2). Indeed, DNase footprinting analysis of a mononucleosome harboring the identical set of nickase recognition sites (Nt.BstNBI) found within nucleosome N5 (12-601-Nt and 12-601Nt/SI) confirmed that these minor sequences variations did not significantly alter the rotational positioning of the DNA relative to the native 601 sequence (Figure S6). Each 12×601 DNA template (Figure 1a) was built on a pUC19 plasmid backbone using a convergent DNA assembly procedure (Figures S1 and S2, Tables S1 and S2), and

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milligram quantities of each plasmid could be readily purified from transformed bacterial cells. Since our immediate downstream goal was to study BER, we chose to incorporate a single dU residue into both 12-601-Nt and 12-601-Nb (Figure 1a). The corresponding plasmid DNAs were digested with their respective nicking endonuclease, followed by addition of a large excess (25-fold) of the corresponding dU modified oligonucleotide designed to replace the original excised fragment (Figure S3 and Table S3). The reaction mixture was subsequently heated at 80°C for 20 minutes, which displaces the shorter DNA fragment from the template and deactivates the nicking enzyme, and then slowly cooled to ambient temperate to ensure proper annealing of the modified strand. The nicks were then resealed by T4 DNA ligase, resulting in generation of a fully intact DNA template. In order to determine the extent of incorporation of the modified oligonucleotide, the 601 DNA fragment containing the modified site was excised from each plasmid and analyzed via an electrophoretic mobility retardation assay (Figures 2b and Figure S3b). The nicked 601 DNA fragment has a small mobility retardation compared to the unnicked DNA, allowing the two products to be easily resolved. The results showed that both nicking and re-ligation reactions proceeded to completion (Figure 2b; lanes 2 and 3, respectively). However, treatment of the DNA with BER proteins UDG and APE1, which generate a single-strand break specifically at dU sites, revealed that a substantial fraction of the DNA template remained unmodified (Figure 2b, lane 4). This suggested that, despite the presence of a large excess of the dU containing oligonucleotide, rehybridization of the unmodified strand was heavily favored during the annealing step.

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We reasoned that this was likely due to formation of a destabilizing mismatch (dU•dC or dU•dG for 12-601-dU49 and 12-601dU88, respectively) upon hybridization of the modified oligonucleotide to the template DNA. We overcame this issue by employing a locked nucleic acid (LNA) capture oligonucleotide (Table S3) during the annealing step, which was designed to sequester the unmodified DNA fragment following its release from the plasmid DNA (Figure 2a). Indeed, when the above exchange procedure was carried out in the presence of a 5-fold excess of capture oligonucleotide, nearly complete incorporation (>95%) of the dU-modified stand was achieved for both DNA templates (Figure 2b; lane 6). Overall, the entire exchange process takes less than 5 hours to complete, allowing rapid access to milligram quantities of modified 12×601 DNA.

Figure 2. Insertion of modified oligonucleotides into 12×601 DNA templates. (a) Schematic representation of the strand exchange process. (b) Representative electrophoretic mobility shift assay demonstrating the insertion of a dU containing oligonucleotide (dU49) into template 12-601-Nt. All reactions were carried out on the corresponding plasmid DNA and the 601 DNA fragment (N5) containing the modified

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site was excised via PflMI and BstXI restriction digestion (Figure 1a) prior to analysis by native polyacrylamide gel electrophoresis.

In addition to single dU residues, we simultaneously incorporated a unique fluorescent nucleotide (Cy3 and Cy5) into each of the two nickable sites within template 12-601-Nt/SI using a single exchange reaction (Figure 1a and Figure S5). Although the use of this template is not discussed further, it demonstrates that our method can be easily extended to allow rapid modification (80%) within the 12-mer array is sequestered within nucleosomes, which effectively reduces the amount of DNA that UDG and APE1 must sample prior to finding the damaged site. Alternatively, compaction of the damaged DNA into a 30 nm fiber, which is expected to increase molecular crowding and effective DNA concentration around the lesion,43 may promote a facilitated diffusion search mechanism by UDG/APE1, thereby increasing the efficiency of lesion identification and repair.58-60 Regardless of the mechanism, these data presented here suggest that densely

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packed chromatin environments (i.e. heterochromatin) may promote the initial steps of BER. Given that BER within linker DNA is not well characterized — this is the first study examining BER of a site-specifically positioned DNA lesion within an oligonucleosome array — it will be important to investigate how the position of a DNA lesion within the linker region, as well as the “linker histone” H1,25,34 affects the activities of BER proteins in the context of different chromatin architectures. Using stochastically damaged 12-mer nucleosome arrays as BER substrates, Nakanishi et al.52 previously reported that the combined activities of UDG and APE1 are only modestly inhibited (~2–3-fold) within compact chromatin. This observation led the authors to conclude that DNA base damage within heterochromatin remains mostly accessible to UDG/APE1, and thus the initial steps of BER do not require significant disruption of chromatin structure. However, the data presented above using sitespecifically modified arrays (Figure 4), which provides a much higher resolution analysis of BER, now suggests otherwise. Indeed, we showed that the rate of uracil removal by UDG/APE1 within compacted nucleosome arrays varied by as much as 70-fold between the two positions tested (Figure 4c). Thus, we conclude that damaged DNA within heterochromatin does not remain mostly accessible to BER initiation, but rather the accessibility varies significantly in a position-dependent manner.

CONCLUSIONS In summary, we have developed a straightforward and efficient method based on nicking endonucleases to generate oligonucleosome arrays containing site-specific DNA modifications. Using this approach, we assembled two different 12-mer nucleosome

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arrays containing site-specifically positioned dU residues, which provided nucleotidelevel resolution of the efficiency of BER within various chromatin environments. A major finding was that inter-nucleosome interactions mediated by histone tail domains (i.e. histone H4) play an important role in regulating BER within higher-order chromatin structures. This implies that modulation of such interactions via histone PTMs, such as lysine acetylation or methylation,61,62 may also regulate BER. Indeed, there is increasing evidence for the involvement of histone modifications in BER pathway.63-66 However, the functional relationships between histone PTMs and BER remain unclear, and only recently have researchers begun addressing these questions using defined in vitro reconstituted systems.67 Therefore, additional work is needed to determine the role of histone PTMs during BER, and the technical approaches reported herein will be useful in that endeavor.

ASSOCIATED CONTENT Supporting information. Supporting Text including Materials and Methods. Table S1– S3 and Figures S1–18. The Supporting Information is available free of charge on the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] Author contributions †These authors contributed equally.

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Notes The authors declare no competing conflict of interest.

ACKNOWLEDGMENTS The authors are grateful to Dr. Wenshe Liu for providing the human histone expression plasmids. This work was supported by the Cancer Prevention and Research Institute of Texas (M1503504).

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