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

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, because of 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 internucleosome interactions play an important role in 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.

Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures

“…S1) 19 . The minor sequence variations resulting from the inclusion of these restriction sites do not significantly alter the rotational positioning of the DNA relative to the native 601 sequence 19 . Treatment of 12-601-Nb with Nb.BbvCI resulted in formation of a short gap upon heat denaturation, which was subsequently filled in with a DNA oligonucleotide carrying the desired dU residue (Table S1).…”

“…We designed the 12 × 601 template ( 12-601-Nb ) to contain two proximal nicking endonuclease recognition sites (Nb.BbvCI) within the nucleosome-bound region of the fifth 601 repeat (N5), which enabled site-specific incorporation of single dU residue using our previously reported “plug-and-play” approach (see “Methods” section and Fig. S1) 19 . The minor sequence variations resulting from the inclusion of these restriction sites do not significantly alter the rotational positioning of the DNA relative to the native 601 sequence 19 .…”

“…In addition, individual nucleosomes are connected by stretches of linker DNA to form chromosome-sized nucleosome arrays that fold and condense into more compact higher-order structures that further impact DNA accessibility and BER 16–18 . For example, the combined catalytic activities of UDG and APE1 are inhibited by as much as 20-fold upon folding of nucleosome arrays into compact 30 nm chromatin fibers 19 .…”

“…Although these studies have provided novel insight into how acetylation potentially regulates BER in vivo , mononucleosomes fall short of being an ideal chromatin model. In particular, they cannot be used to assess the impact of histone acetylation (or other PTMs) on inter-nucleosomal interactions and higher-order chromatin folding, which are likely key factors that regulate BER at the chromatin level 19,32 .…”

Nucleosomes and the three glycosylases: High, medium, and low levels of excision by the uracil DNA glycosylase superfamily

“…The biochemical activities of these glycosylases have been characterized in duplex DNA, however there have been fewer examinations of UDG and SMUG1 activity on chromatin or packaged DNA (36-40). The studies on packaged DNA have revealed that UDG and SMUG1 activity is generally lower than on duplex, and that local dynamics of the DNA and associated proteins can modulate activity.…”