Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

Article

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease Graphical Abstract

Authors Soo-Mi Kweon, Bing Zhu, Yibu Chen, L. Aravind, Shuang-Yong Xu, Douglas E. Feldman

Correspondence [email protected]

In Brief Kweon et al. uncover a function for the SRAP domain, which couples autoproteolytic cleavage to activation of a nuclease selective for DNA containing Tet-oxidized derivatives of 5-methylcytosine. These findings reveal a mechanism for targeted erasure of DNA methylation via the stepwise enzymatic actions of Tet and SRAP.

Highlights d

The ancient SRAP domain acts as an autopeptidase-coupled nuclease

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SRAP nucleases selectively cleave DNA containing Tetoxidized 5-methylcytosine

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Inactivation of murine Srap1 results in accumulation of 5hmC in genomic DNA Srap1 deficient mice display embryonic sublethality and altered DNA methylation

Kweon et al., 2017, Cell Reports 21, 482–494 October 10, 2017 ª 2017 The Author(s). https://doi.org/10.1016/j.celrep.2017.09.055

Data and Software Availability GSE81222

Cell Reports

Article Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease Soo-Mi Kweon,1 Bing Zhu,1 Yibu Chen,2 L. Aravind,3 Shuang-Yong Xu,4 and Douglas E. Feldman1,5,* 1Department

of Pathology Service, Department of Health Sciences Libraries University of Southern California, Keck School of Medicine, Los Angeles, CA 90033, USA 3NCBI, NIH, Bethesda, MD 20894, USA 4New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2017.09.055 2Bioinformatics

SUMMARY

Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development. INTRODUCTION Enzymatic methylation of cytosine at the C5 position (5mC) is among the most phyletically conserved modifications of DNA and constitutes a stable and heritable form of gene control €beler, 2015; Smith and Meissner, 2013; Ziller et al., (Schu 2013). In mammals, erasure of 5mC, most often in the context of CpG dinucleotides, underlies reprogramming of parental genomes to the totipotent state upon fertilization and, later in development, germline cell maturation through the removal of gene imprinting (Smith et al., 2012; Wang et al., 2014; Yamaguchi et al., 2013; Gkountela et al., 2015). Distinct mechanisms underlie DNA demethylation during these key developmental transitions. Nuclear exclusion of an embryonically expressed isoform of the maintenance methyl-

transferase Dnmt1 and its obligate partner UHRF1, in conjunction with successive rounds of DNA replication and cell division, results in dilution of 5mC in daughter cells (Inoue and Zhang, 2011; von Meyenn et al., 2016). Enzymatic oxidation of 5mC by the Tet family of dioxygenases (Tet1-3) reinforces this process, as the most abundant Tet oxidation product, 5-hydroxymethylcytosine (5hmC), repels Dnmt1 (Hashimoto et al., 2012), blocking methylation of newly synthesized DNA strands. Individual CpGs are also subject to rapid and complete demethylation in the absence of intervening rounds of DNA replication (Guo et al., 2014a, 2014b; Wang et al., 2014). Iterative oxidation of 5hmC generates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), which are then removed from DNA through the actions of thymine DNA glycosylase (TDG) and the base excision repair (BER) pathway (He et al., 2011; Shen et al., 2013; Weber et al., 2016). However, the relative scarcity of 5fC and 5caC and the unimpaired demethylation of paternal pronuclear DNA in Tdg-deficient embryos together implicate additional factors in the removal of CpG methylation (Guo et al., 2014a; Song et al., 2013). 5hmC and 5fC act not only as intermediates in demethylation but as stable, locally abundant marks that may affect the interaction of diverse transcriptional regulators with DNA (Bachman et al., 2014; Kriaucionis and Heintz, 2009; Yin et al., 2017); how these alternative fates are specified is not yet understood. In this work, we explore the function of the experimentally uncharacterized protein C3ORF37/Hmces, identified as a selective reader of Tet-oxidized derivatives of 5mC, but not 5mC, in mouse embryonic stem cell extracts (Spruijt et al., 2013), suggesting its involvement in interpreting or processing such marks. C3ORF37/Hmces (referred to hereafter Srap1) represents the only known mammalian ortholog of the SRAP domain superfamily, which is widely distributed across bacteria and bacteriophage phyla and is operonically associated with the genetic response to sustained DNA damage in these systems (Aravind et al., 2013). We show that the SRAP domain catalyzes removal of 5hmC and its further oxidized derivatives from genomic DNA through an autopeptidase-coupled, modification-selective endonuclease that incises across DNA sites containing Tetoxidized bases. These findings uncover an essential, fully enzymatic route for the targeted erasure of DNA methylation during embryogenesis.

482 Cell Reports 21, 482–494, October 10, 2017 ª 2017 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 1. Autoproteolytic Cleavage by the SRAP Domain (A) Diagram of SRAP protein orthologs from E. coli (YedK) and mouse (Srap1) showing the percentage amino acid identity and similarity between SRAP domains. The positions of the autopeptidase active site cysteine (C), glutamate (E), and histidine (H) residues are shown. Red arrow indicates proposed cleavage site N-terminal to the catalytic cysteine. (B) Full-length Srap1 (1–353) or the indicated truncations, each containing an N-terminal Flag-HA (FHA) tag, was expressed in HEK293T cells. Samples were collected 2 days after transfection and analyzed by SDS-PAGE and immunoblotting. Cleavage of the FHA tag generates products of lower molecular mass (lanes 1 and 2). Tubulin, loading control. Results are representative of n = 4 experiments. (C) Wild-type (WT) or autopeptidase active site mutants (C2A, H209Q) of 3xFlag-SRAPd-HA was expressed in HEK293T cells and detected with anti-Flag or antiHA antisera. The mean percentages of tag cleavage with SD (n = 5 experiments) are shown below each lane. (D) Purified, recombinant wild-type and DC2 mutant variants of SRAPd were resolved by SDS-PAGE and Coomassie blue staining. A small amount of Hsp60/70 chaperones is bound to each recombinant protein. (E) In vitro autopeptidase assay. Wild-type or DC2 mutant SRAPd was incubated for the indicated times at 37 C in the absence or presence of DNA duplex oligo containing a single 5hmC in a CpG sequence context. Following incubations, SRAPd was resolved by SDS-PAGE and immunoblotting using anti-Srap1 antisera. A schematic diagram of recombinant SRAPd is shown below. (F) Scaled sequence logo diagram of the C-terminal extension present in metazoan SRAP domain proteins. The size of each amino acid residue is proportional to its propensity. Numbers show amino acid positions. (G) FHA-Srap1 or the indicated point mutants was expressed in HEK293T cells and detected with anti-HA or anti-Srap1 antisera. Shown are the mean percentages of tag cleavage with SD (n = 3 experiments). See also Figure S1.

RESULTS Autoproteolytic Cleavage of the SRAP Domain In DNA pull-down assays, purified recombinant glutathione S-transferase (GST)-Srap1 fusion protein bound stably to DNA duplex oligonucleotides with a single CpG site containing 5hmC, 5fC, or 5caC but was barely detectable in association with 5mC, indicating a direct and selective association with Tet-oxidized derivatives of 5mC (Figures S1A–S1C). SRAP domains possess an invariant cysteine at position 2 (Cys2) along with exceptionally conserved glutamate and histidine residues (Figure 1A), proposed to constitute a catalytic triad for a thiol autopeptidase (Aravind et al., 2013). We expressed in HEK293T cells full-length murine Srap1 or truncations encompassing either the standalone SRAP domain (SRAPd; residues 1–240)

or the C-terminal extension present in metazoan SRAP orthologs (residues 240–353), each containing an amino-terminal Flaghemagglutinin (FHA) double tag. In contrast to full-length Srap1 and the C-terminal extension, SRAPd was not detected with Flag antisera (Figure 1B). Mutation of either Cys2 to alanine (C2A) or the conserved histidine to glutamine (H209Q) in SRAPd led to accumulation of an N-terminal 3xFlag tag (Figure 1C). Comparison of Flag epitope levels between wild-type and the autopeptidase mutants indicated that SRAPd exists predominantly (
90%) in its cleaved form, with a molecular mass of
3 kDa less than that of autopeptidase inactive variants, as determined by immunoblot against a C-terminal hemagglutinin (HA) epitope tag on the same construct (3xFlag-SRAPd-HA) (Figure 1C). These observations support the proposal that autoproteolytic cleavage releases residues N-terminal to the catalytic

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cysteine. Highly purified, recombinant SRAPd, but not a mutant lacking Cys2 (DC2), likewise underwent proteolytic cleavage when incubated at 37 C; cleavage proceeded independently of 5hmC-containing DNA added to the reaction (Figures 1D and 1E). The C-terminal extension contains conserved tryptophanleucine (WL) motifs (Figure 1F), which might sterically occlude enzyme active sites. Mutation of one such motif to glycinealanine (W289G/L290A) stimulated cleavage of the N-terminal tag in FHA-Srap1 expressed in HEK293T cells, while the further introduction of an autopeptidase-inactivating mutation (H209Q) reversed this effect (Figure 1G), indicating an autoinhibitory role for the C-terminal extension. Srap1 Promotes Turnover of Tet-Oxidized 5mC in Genomic DNA Wild-type and autopeptidase-inactive variants of SRAPd stably bound to chromatin in HEK293T cells (Figure S1D) and to resin coated with DNA duplex oligonucleotide containing 5hmC or 5caC (Figure S1E), indicating that autoproteolytic cleavage is dispensable for DNA binding. To probe Srap1 function, we coexpressed the catalytic domain (CD) of HA-tagged Tet1 (HATet1-CD) with either FHA-Srap1 or the wild-type or autopeptidase inactive (C2A) variants of 3xFlag-SRAPd-HA. Whereas HA-Tet1-CD increased levels of 5hmC, full-length Srap1 and wild-type SRAPd, but not the C2A mutant, each reversed this effect, as determined by dot blot assay of genomic DNA, with corresponding decreases in the abundance of 5fC (data not shown) and 5caC detected in the same samples (Figure 2A). These observations implicate the SRAP domain in turnover of genomic 5hmC and its oxidized derivatives to unmodified cytosine. Srap1 mRNA was detected in mouse ESCs at levels comparable with Tet1 and Tet2 transcripts, as determined by RNA sequencing (RNA-seq) analysis (Figure S2A), with lower expression in embryonic day (E) 9.5 embryos and in most adult organs, as determined by qRT-PCR (Figure S2B), raising the possibility that Srap1 might operate on Tet-oxidized bases in ESCs (Hon et al., 2014; Pastor et al., 2011). To investigate Srap1 function, we applied CRISPR/Cas9-mediated gene targeting in mouse zygotes (Wang et al., 2013) to generate deletions in the first protein coding exon of Srap1. PCR genotyping of the resulting founder mice and Sanger sequencing of product amplicons identified two alleles, which were carried forward for further studies: deletions of 388 bp (D388) or 71 bp (D71), each ablating exonic sequences encompassing the translation initiation codon and catalytic Cys2 for predicted knockout (KO) alleles (Figure S3). Biallelic Srap1 KO and wild-type ESCs co-derived from intercrosses of D71 and D388 heterozygous mice exhibited typical, flattened colony morphology, although colonies composed of Srap1 KO cells were less rounded than wildtype colonies (Figure S4A). Prominent nuclear and perinuclear Srap1 immunoreactivity was detected in wild-type but not Srap1 KO cells (Figures S4B). Transcript levels of DNA cytosine methyltransferases (Dnmt1/3a/3b), pluripotency-associated transcription factors (Pou5f1/Oct4, Sox2, Nanog), Tdg, and Tet genes (Tet1-3) were not significantly altered in Srap1-deficient cells, as determined by qRT-PCR (Figure S4C), and no effect was seen on the rate of cell proliferation in vitro (Figure S4D).

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We then performed quantitative ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analysis of genomic DNA hydrolysates prepared from wild-type and Srap1 KO ESCs, as well as Srap1 KO ESCs stably expressing FHA-Srap1 (Figure 2B). These studies revealed a global accumulation of Tet-oxidized 5mC derivatives, relative to total deoxycytidine (dC), in Srap1 KO cells, with the largest increases detected for 5fC and 5caC (2.65- and 3.36-fold, respectively), while 5hmC levels were 1.73-fold greater (0.195% versus 0.115%). 5mC levels also increased in Srap1 KO cells, suggesting an indirect effect of impaired 5hmC/5fC/5caC processing (Figures 2C and 2D). Reintroduction of Srap1 in Srap1 KO cells restored levels of 5mC and its Tet-oxidized derivatives to those of wild-type ESCs (Figure 2D). Short hairpin RNA depletion of Tdg further increased the relative abundance of 5fC and 5caC in Srap1 KO cells, above levels detected in wild-type ESCs depleted of Tdg (Shen et al., 2013), while no effect was seen on 5hmC (Figures 2E and 2F). Thus, TDG removes a subset of ectopic 5fC and 5caC, but not 5hmC, from genomic DNA in Srap1 KO cells. The SRAP Domain Contains a Modification-Selective Nuclease In contrast to reactions with TDG, glycosylase activity was not detected in reactions of purified Srap1 or SRAPd with DNA substrate containing 5caC (data not shown). We then considered a possible nuclease function for SRAP, given both the recurrent involvement of endo- and exonucleases in the repair of DNA damage and the operonic association of prokaryotic SRAP orthologs with genes implicated in the SOS response (Aravind et al., 2013; Deshpande et al., 2016; Smogorzewska et al., 2010). To test this, we first incubated PCR products fully substituted with 5mC, 5hmC, 5fC, or 5caC, or containing unmodified cytosine, in the presence of purified GST-Srap1. Analysis of reaction products by gel electrophoresis revealed extensive degradation of modified DNA containing 5hmC or its further oxidized derivatives, whereas DNA containing 5mC or unmodified cytosine was resistant to digestion by Srap1 (Figure 3A). Highly purified recombinant YedK, but not a DC2 mutant, also selectively degraded 5hmC-containing DNA substrate (Figures 3B and 3C). To map Srap1 cleavage sites, we performed runoff sequencing of partially digested DNA substrate containing 5hmC in place of cytosine. These studies revealed efficient cleavage immediately 30 of 5hmC in a CpG sequence context, with incisions also detected at CpT dinucleotides (Figures 3D and 3E). An additional cleavage product was detected 50 of the major cut site at CpG, suggesting secondary exonuclease processing (Figure 3D). We next challenged purified SRAPd or YedK with a 50 fluorescein amidite (FAM)-labeled DNA duplex containing a single 5hmC within a CpG sequence context. Analysis of reaction products by native PAGE revealed cleavage in close proximity to 5hmC, in agreement with the findings of the runoff sequencing studies and indicating that the SRAP domain can cleave substrate containing a single 5hmC (Figure 3F). As with YedK, murine SRAPd lacking the catalytic Cys2 was inactive (Figure 3G). Duplex DNA containing a single 5caC was also cleaved by SRAPd (Figure 3G). Denaturing PAGE analysis

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Figure 2. Srap1 Promotes Erasure of Tet-Oxidized 5mC from Genomic DNA (A) Dot blot analysis of 5hmC and 5caC levels in genomic DNA. Empty vector (Empty) or plasmids expressing FHA-Srap1 or WT or autopeptidase-inactive mutant (C2A) variants of 3xFlag-SRAPd-HA were co-transfected with HA-Tet1-CD into HEK293T cells. 5hmC and 5caC in serially diluted genomic DNA was detected using the respective antisera. The mean signal intensities with SD from n = 3 experiments are shown below each lane. Methylene blue shows total membranebound DNA. HA-Tet1-CD was detected by anti-HA immunoblotting of cell extracts. FHA-Srap1 and SRAPd were detected by Srap1 antisera. (B) Immunoblot of Srap1 in lysates prepared from WT and Srap1 KO ESCs, in the absence or presence of stably expressed FHA-Srap1 as indicated. Cleavage of the N-terminal FHA tag generates a product of lower molecular mass (lane 3). A redundant lane was removed. Image is representative of n = 2 experiments. (C) Overlaid extracted ion chromatograms of digested genomic DNA from WT and Srap1 KO mouse ESCs. The MS signal intensities of each cytosine derivative were normalized to the deoxycytidine (dC) content of WT cells.

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of products generated in extended reactions of SRAPd with 5hmC-containing substrate revealed 30 /50 exonuclease processing extending from 5hmC (Figure 3H). These data together indicate that the SRAP domain catalyzes nucleolytic cleavage of DNA across sites containing Tet-oxidized 5mC, suggesting that strand resynthesis across cleaved sites might enable replacement of 5hmC with unmodified cytosine. In support of this proposal, we reconstituted the substitution of 5hmC with unmodified cytosine in the presence of Srap1, DNA polymerase I, dNTPs and T4 ligase, as determined by restoration of an HpaII-sensitive restriction site (Figure 3I). Genomic Distribution of Srap1 in Mouse ESCs To examine the genomic distribution of Srap1, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analysis of FHA-Srap1 expressed in mouse ESCs and identified 2,824 high-confidence peaks (false discovery rate [FDR] < 0.05, 5.37fold mean enrichment of Srap1 signal relative to IgG control; Tables S1 and S2), a fraction of the total 5hmC- or 5fC-marked regions identified in ESCs through similar affinity-enrichment strategies (Pastor et al., 2011; Song et al., 2013; Xu et al., 2011). Compared with the same number of size-matched, random DNA fragments shuffled throughout the genome, Srap1-bound peaks significantly overlapped with 5hmC- and 5fC/5caC-containing CpGs identified through base-resolution mapping studies (Wu et al., 2014; Yu et al., 2012) (124.9 and 179.7 times more peaks, respectively, than expected by chance) (Figure 4A; Table S3). Srap1-bound sites were enriched within gene promoters (1.7-fold enrichment within 0.5 kb of TSS; 3.8-fold enrichment within 1.0 kb of TSS) and in transcribed regions of gene bodies, including in 50 and 30 UTRs (Figure 4B), as also observed for the 5hmC mark (Pastor et al., 2011; Williams et al., 2011). Intergenic Srap1 targets (n = 1,866) were enriched in L1 long interspersed nuclear elements (LINEs) and, to a lesser extent, in B1-Alu and B2 classes of short interspersed nuclear elements (SINEs) (Figure 4C), repeat elements that undergo demethylation in preimplantation embryos (Shen et al., 2013; Smith et al., 2012). Srap1 also bound to a subset of class II endogenous retroviruses (ERVs), consistent with the presence of 5hmC and 5fC that accompanies limited demethylation of these elements (Figure 4C) (Shen et al., 2013; Walsh et al., 1998). Detailed analysis of Srap1 read density plotted across a metagene revealed a moderate depletion approaching the transcription start site (TSS) followed by a continuous increase along the gene body toward the transcription end site (TES), after which Srap1 density decreases sharply. Upstream of the TSS, Srap1 read density is relatively elevated in the top 10% of highly expressed genes and lower in the bottom 10% of least expressed genes, whereas along exonic sequences of the gene body, this pattern is reversed (Figure 4D). The distribution of Srap1 parallels

that of 5hmC in genes linked to high-density CpG promoters and at gene promoters containing the univalent H3K4me3 histone mark, associated with active transcription (Shen et al., 2013; Xu et al., 2011). We next compared the distribution of Tet-oxidized 5mC derivatives in wild-type and Srap1 KO ESCs by DNA immunoprecipitation (DIP) sequencing (DIP-seq) using modification-specific antisera. Srap1 deficiency resulted in a global increase in the number of called 5hmC- and 5fC/5caC-enriched regions (empirical FDR < 0.1) (Figure 4E; Table S4), substantiating the findings of the LC-MS/MS analysis. A subset of 5hmC peaks in Srap1 KO cells (23.7% [n = 142,146]) did not overlap those detected in wild-type cells, and most 5fC/5caC peaks (80.0% [n = 13,154]) also mapped uniquely in KO cells. A majority of called Srap1 peaks (1,948 [69.0% of Srap1 peaks]) overlapped with ectopic 5hmC sites, while a smaller fraction (964 [34.1% of Srap1 peaks]) overlapped with ectopic 5fC+5caC (Figure 4E), underscoring the role of Srap1 in turnover of a subset of these Tet-oxidized bases while suggesting differences in reaction or dissociation rates at each species of modified base in vivo. Applying a more stringent cutoff (FDR < 0.02), we identified 3,254 5hmC peaks in Srap1 KO cells (Table S4), of which 97.4% were unique to KO cells. Srap1 deficiency affected 5hmC distribution across a metagene, with KO-specific increases approaching the TSS in highly expressed genes and along the gene body in genes expressed at low levels, whereas 5mC and 5caC levels were not significantly changed in these regions (Figures 4F–4I), in agreement with the role of TDG in removal of 5fC/5caC at gene promoters (Neri et al., 2015). Srap1 deficiency also increased the density of 5hmC and 5caC across the majority of H3K4me1+ enhancer elements (Figure 4J). RNA-seq analysis identified 224 genes that displayed altered expression in Srap1 KO ESCs (log2 fold change [FC] > 2.0), with 98 upregulated and 126 downregulated genes (Figure 4K; Table S5). Gene ontology analysis of downregulated genes in Srap1 KO ESCs revealed involvement in developmental processes and cell differentiation (Figure 4L). qRT-PCR validation confirmed that transcript levels of Pou3f1, Zscan4c, and Sox3, key determinants of developmental potency and neural cell fate specification in early embryos (Amano et al., 2013; Nishimura et al., 2012; Zhu et al., 2014), are aberrantly downregulated in the absence of Srap1 (Figure 4M). Embryonic Sublethality in Srap1-Deficient Mice Although Srap1 KO ESCs were recovered at the expected frequency from intercrosses of Srap1 D388 and D71 heterozygous mice (4 of 11 clones tested), the combined genotype frequency distribution in mid- to late gestation embryos (E9.5–E16.5) and postnatal pups (n = 190 from 21 litters) deviated from the expected Mendelian ratio, with significantly fewer KO progeny (Figures 5A and S5A). Srap1 KO embryos (n = 11 from 7 litters) were

(D) Levels of cytosine derivatives relative to total dC content in WT and Srap1 KO ESCs, in the absence or presence of stably expressed FHA-Srap1, as determined by quantitative UHPLC-MS/MS. Data are plotted as mean with SD from n = 3 experiments. **p < 0.01, two-tailed t test. (E) Lysates prepared from WT (lanes 1 and 2) and Srap1 KO (lane 3) ESCs, in the absence or presence of lentiviral short hairpin RNA (shRNA) depletion of Tdg (shTdg), were resolved by SDS-PAGE and immunoblotting using anti-TDG antisera. Tubulin, loading control. Images are representative of n = 2 experiments. (F) Levels of cytosine derivatives relative to total dC content in ESCs of the indicated genotypes, in the absence or presence of sh-Tdg, determined by quantitative UHPLC-MS/MS. Data from Srap1 ESCs are the same as shown in (D). All data show mean ± SD from n = 3 experiments. **p < 0.01, two-tailed t test. See also Figures S2–S4.

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Figure 3. The SRAP Domain Contains a Modification-Selective Nuclease (A) In vitro DNA endonuclease assay. PCR products containing unmodified cytosine (C) or fully substituted with 5mC, 5hmC, 5fC, or 5caC were incubated with buffer alone, GST-Srap1, or restriction endonuclease HpaII, which cleaves recognition sites containing unmodified cytosine and generates a major product of 735 bp. Reaction products were resolved by nondenaturing agarose gel electrophoresis. Images are representative of n = 3 experiments. (B) Purified wild-type and DC2 mutant variants of YedK used in nuclease assays. Recombinant proteins were purified from bacterial lysates and resolved by SDSPAGE and Coomassie blue staining.

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morphologically normal (Figures 5B and S5B), and we did not observe empty implantation sites or absorbed KO conceptuses in utero. Some Srap1+/ heterozygous embryos, including progeny from Srap1 KO crossed to wild-type, failed to develop properly and were found as a mass of highly vascularized, undifferentiated tissue (3.9% [n = 155 Srap1+/ heterozygotes from 26 litters]; 0.0% [n = 56 wild-type from 21 litters]) (Figure S5C). Srap1 KO pups were underweight at 3 weeks of age (Figure S5D) but developed normally into fertile adults. In-crossed Srap1 KO mice produced smaller litters than intercrossed Srap1+/ heterozygotes in both C57BL/6N and129/Sv strain backgrounds (Figure 5C), but in-cross progeny were morphologically normal (n = 184 from 27 litters) (Figure 5D). Thus, Srap1 deficiency causes a sublethal phenotype manifested in late blastocysts, prior to implantation (Papaioannou and Behringer, 2012). The apparent timing of this sublethality is consistent with deregulated expression of genes that act in the control of developmental potency in early implantation stage embryos (Figure 4M) (Amano et al., 2013; Nishimura et al., 2012; Zhu et al., 2014). Aberrant Patterns of DNA Methylation in Srap1 KO Embryos To examine the impact of Srap1 deficiency on the DNA methylome, we performed reduced representation bisulfite sequencing (RRBS) (Meissner et al., 2005), generating base resolution maps of 5mC+5hmC in CpG-enriched regions from genomic DNA of E9.5 wild-type and Srap1 KO littermates. Srap1 KO embryos exhibited a global increase in mean 5mC+5hmC levels (53.6% in wild-type controls versus 58.8% in Srap1 KO) (Table S1) and an altered pattern of DNA methylation at individual cytosines, with an increased fraction of highly methylated (>70% methylation) CpGs (Figure 6A). We identified 1,589 differentially methylated regions (DMRs) (median 5mC+5hmC level: 1.5% in wild-type versus 23.5% in Srap1 KO) (Figure 6B), of which 1,457 displayed >10% change in methylation (1,292 hypermethylated, 165 hypomethylated) in Srap1 KO, as well as 1,157 unique genes with promoter CpG island (CGI) methylation changes (Table S6). Alignment of DMRs exhibiting larger absolute changes in methylation (>20% 5mC+5hmC change level, n = 785) with annotated genomic elements revealed a net increase of 5mC+5hmC at proximal promoters (n = 521) and

enhancer elements (n = 29) (Figure 6C; Table S6). Genes with increased 5mC+5hmC at promoter CpG islands are functionally associated with cell fate commitment and tissue formation (Figure S5E). Compared with the same number of size-matched DNA fragments distributed randomly throughout the genome, significantly more E9.5 DMRs than expected by chance overlapped or were proximal to (<1.5 kb) ectopic 5hmC peaks identified in Srap1 KO ESCs using stringent cutoff criteria (ectopic 5hmC peaks: 21 of 1,589 DMRs, o/e = 5.4; all 5hmC peaks: 295 of 1,589 DMRs, o/e = 1.6) (Figures 6D and 6E). Although 5hmC can act as an intermediate in DNA demethylation (Dawlaty et al., 2013; Yamaguchi et al., 2013), DMRs corresponding to sites with ectopic 5hmC in ESCs displayed a range of methylation changes, including both hypo- and hypermethylation (Figures S5F and S5G). We performed ChIP-qPCR to validate Srap1 binding at two such DMRs: the Fbxo33 gene promoter and Nespas/Gnas locus (Figure 6F, top). DIP-qPCR analysis confirmed accumulation of 5hmC at these sites in Srap1 KO ESCs (Figure 6F, bottom). Both DMRs were linked to aberrant expression of associated genes in individual E9.5 Srap1 KO embryos, as determined by qRT-PCR (Figure 6G). E9.5 DMRs were also significantly associated with genes that displayed differential expression in Srap1 KO ESCs (n = 11 of 224 genes) (Figure S5H). Thus, Srap1 inactivation results in aberrant patterns of DNA methylation associated with deregulated gene expression in both ESCs and mid-gestation embryos. DISCUSSION Sweeping erasure of DNA cytosine methylation transforms parental genomes upon fertilization and underlies reprogramming to totipotency. A combination of passive demethylation through replicative dilution and, through a poorly understood mechanism, the rapid and targeted removal of 5mC are thought to drive this process (Inoue and Zhang, 2011; Guo et al., 2014a; Wang et al., 2014; Peat et al., 2014). In this work, we uncover a role in DNA demethylation for the SRAP domain protein Srap1, the mammalian ortholog of a previously unknown class of DNA base modification-selective nucleases widely distributed across bacteria, bacteriophage, and eukaryotic phyla. The SRAP domain couples autoproteolytic cleavage to nuclease activation

(C) PCR products containing unmodified cytosine (C) or fully substituted with 5mC or 5hmC were incubated with buffer only or the wild-type or DC2 mutant variants of YedK. Reaction products were resolved by nondenaturing agarose gel electrophoresis and imaged by SYBR Gold staining (n = 3 experiments). (D) Sanger sequencing of DNA digestion products. DNA substrate fully substituted with 5hmC in place of cytosine was generated by PCR, then digested with Srap1 or 5hmC-selective endonuclease KpnXI. Products were purified and sequenced. Arrows indicate major cleavage sites, denoted by the presence of an ectopic adenine (A). Shaded blue box shows CpT cleavage site, shaded gray box indicates additional KpnXI cleavage site. Red asterisk denotes secondary Srap1 incision product. (E) DNA sequence LOGO diagram of Srap1 cleavage sites identified by runoff sequencing. Black arrow indicates position of DNA strand cleavage. (F) In vitro DNA endonuclease assay. Duplex DNA labeled on the top strand with a 50 FAM and containing a single CpG site with 5hmC was incubated in buffer only, SRAPd or YedK, or the restriction endonuclease MslI or FatI for 1 hr. A schematic map of DNA substrate is shown at top. Reaction products were resolved by native PAGE and fluorescence imaging (n = 3 experiments). (G) Top strand 50 FAM-labeled 32-mer DNA substrate containing a single 5hmC or 5caC was incubated in buffer alone or in the presence of WT or DC2 variants of SRAPd. Products were resolved by denaturing PAGE and fluorescence imaging. Results are representative of n = 4 experiments. (H) Duplex DNA labeled on the top strand with 50 FAM and with a single CpG site containing 5hmC (2.5 mM) was incubated in extended reactions (3 hr) with SRAPd or the indicated restriction enzymes. Products were resolved by denaturing PAGE and fluorescence imaging. M, size marker (n = 3 experiments). (I) Reconstitution of enzymatic 5hmC turnover to cytosine. DNA duplex containing unmodified cytosine or a single 5hmC was incubated in the absence or presence of Srap1 or the indicated factors for 1.5 hr, recovered by spin column purification, and subjected to digestion by HpaII. Products were resolved by denaturing PAGE analysis (n = 3 experiments).

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and DNA strand incision at sites containing 5hmC and its Tetoxidized derivatives, revealing a mechanistic framework for the targeted erasure of DNA cytosine methylation through the stepwise enzymatic actions of Tet and SRAP. Specialized DNA exo- and endonucleases are deployed in prokaryotes and mammalian cells to counteract potentially life-

Figure 5. Embryonic Sublethality in Srap1 KO Mice (A) Genotypic analysis of progeny from intercrossed Srap1 ± heterozygous (Het) mice in C57BL/6N strain background, showing predicted and observed frequencies for each genotype. P value, chi-square test. (B) Representative image of E13.5 embryos recovered from a single litter of intercrossed Srap1 ± mice. Red asterisk indicates Srap1 KO embryo. Scale bar represents 5 mm. (C) Mating scores of intercrossed Srap1 ± heterozygotes and in-crossed Srap1 KO mice in C57BL/ 6N or 129/Sv strain backgrounds. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by cesarean section. Red lines indicate the mean litter size. P value calculated by two-tailed t test. (D) Neonatal Srap1 KO pups from a single litter of in-crossed Srap1 KO parents. Scale bar represents 10 mm. See also Figures S3 and S5.

threatening DNA damage, including cyclobutane pyrimidine dimers, interstrand crosslinks, and protein-DNA adducts that could stall replication (Deshpande et al., 2016; Smogorzewska et al., 2010). Diverse modification-dependent nucleases have been identified in bacteria, including those that selectively cleave DNA containing 5hmC (Borgaro and Zhu, 2013). SRAP enzymes,

Figure 4. Srap1-Dependent Turnover of 5hmC at Genomic Elements (A) The number of Srap1-bound peaks or random DNA fragments of similar size shuffled throughout the genome (n = 2,824), or control IgG peaks (n = 113), containing at least one called 5hmC (left) or 5fC/5caC (right). Red numbers on the top indicate the ratio between observed and random. ***p < 0.001, Fisher’s exact test. (B) Diagram illustrating the distribution of Srap1-bound regions into promoter (1 to +1 kb relative to TSS), 50 and 30 UTR, exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of Srap1 at each feature. (C) Relative enrichment of Srap1-bound DNA fragments within each class of repetitive element in mouse ESCs. (D) Normalized tag density of Srap1 peaks plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (red) or the bottom 10% of least expressed genes (blue). IgG control tag density is represented as gray trace. (E) Venn diagrams showing the relative distribution of 5hmC or 5fC+5caC peaks in WT and Srap1 KO mouse ESCs. Purple shaded circles represent called Srap1 peaks (n = 2,824). (F) Normalized 5hmC read density distribution 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES in WT and Srap1 KO cells. Color-coded plots show 5hmC distribution across genes stratified by expression level. Isotype-matched IgG control tag density traces from WT and Srap1 KO are shown for comparison. (G) Heatmap representation of normalized 5hmC and 5caC read densities centered on a window ± 2 kb relative to the TSS. Heatmaps are rank-ordered by the mean of each signal in WT cells. (H) Normalized tag density of 5mC, 5hmC and 5caC centered at transcription start sites (TSS) in WT and Srap1 KO cells. (I) Genome browser view showing 5hmC/5fC/5caC distributions in control and Srap1-deficient ESCs at a representative locus. Shaded vertical bars highlight promoter regions containing ectopic 5hmC. RefSeq exon structure (blue) for each gene is shown at the bottom. (J) Heatmaps of Srap1, and 5hmC and 5caC at H3K4me1+ enhancer elements in WT and Srap1 KO cells, rank-ordered by the mean of each signal in WT cells. (K) Scatterplot presentation of log2 FPKM values for genes expressed in WT and Srap1 KO ESCs as determined by RNA-seq. Genes significantly up- or downregulated (log2 FC > 2.0, p < 0.005, Fisher’s exact test) are indicated in blue. The gray diagonal is provided for reference. (L) Gene Ontology analysis of 126 downregulated genes in Srap1 KO cells showing that most genes are implicated in embryonic development and cell differentiation. The yellow vertical line indicates the threshold for significance. (M) qRT-PCR analysis of relative transcript levels for the indicated genes in WT and Srap1 KO ESCs. Expression is normalized to Gapdh levels in each sample. Error bars indicate SEM for n = 3 experiments. See also Tables S1, S2, S3, S4, and S5 and Figures S2 and S3.

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Figure 6. Impact of Srap1 Deficiency on the DNA Methylome (A) Distribution of DNA methylation levels in WT and Srap1 KO embryos at E9.5 determined by RRBS. The x axis represents CpG methylation levels binned in increments of 10%. (B) Boxplot of CpG methylation levels at differentially methylated regions (DMRs) (n = 1,589), defined as regions containing significant (p < 0.01, false discovery rate [FDR]-adjusted p value, chi-square test) changes in methylation. Black lines in the colored boxes indicate the median, box edges indicate the 25th and 75th percentiles, and whiskers the 0th and 100th percentiles. P value, paired ANOVA multiple-comparison test. (C) Distribution of DNA methylation changes (Srap1 KO-WT; cutoff 20%) at annotated sequence elements. Change levels are binned in increments of 20%. TES, transcription end site. (D) The number of Srap1-dependent DMRs, or random DNA fragments of similar size, proximal to (<1.5 kb) one or more ectopic 5hmC peaks called in Srap1 KO ESCs. Red number indicates the ratio between observed and random. P value, Fisher’s exact test. (E) Genome browser views of representative loci showing Srap1-bound sites and 5mC/5hmC/5fC/5caC peaks in WT and Srap1 KO ESCs. Shaded vertical bars highlight regions containing ectopic 5hmC. RefSeq exon structure (blue) and location of CpG islands (green) are shown below. Bottom, DNA methylation profiles from RRBS analysis of E9.5 embryos showing the methylated (5mC+5hmC) fraction for each cytosine scored, the mean percentage of methylation across each DMR with SD (n = 2 experiments), and 5mC+5hmC change levels (Srap1 KO-WT). (F) ChIP-qPCR (top) or DIP-qPCR (bottom) analysis for the Nespas/Gnas and Fbxo33 loci in WT and Srap1 KO ESCs. The indicated antisera were used for immunoprecipitation. The average value for each condition (n = 2 experiments) is shown. Error bars indicate SEM. (G) qRT-PCR analysis of gene expression patterns in E9.5 WT controls and individual Srap1 KO embryos. Expression is normalized to Gapdh levels in each sample. The average value of control embryos (n = 3) is set as 1. Error bars indicate SEM. See also Tables S1 and S6 and Figures S3 and S5.

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however, possess an autopeptidase-coupled nuclease, adding an additional step and potential point of control to its nuclease activity. We propose that this combination of enzymatic properties conferred a unique advantage to the SRAP superfamily that was exploited for targeted removal of DNA cytosine methylation. Notably, transient, Tet3-dependent DNA lesions are detected concurrent with demethylation of pronuclear DNA (Ladsta¨tter and Tachibana-Konwalski, 2016). Thus, tight control of both Srap1 and TDG, which removes 5fC/5caC from DNA in concert with the APE1 endonuclease (Weber et al., 2016), may be critical to restrict these potentially deleterious DNA strand breaks. Our analysis of Srap1-deficient ESCs and embryos reveals that highly specific removal of Tet-oxidized 5mC by Srap1 sustains DNA methylation homeostasis during embryogenesis. Srap1-deficient ESC genomes display an increase in both 5hmC and 5fC/5caC peaks (Figure 4E), with ectopic peaks often found in close proximity to other 5hmC or 5fC/5caC sites that are unaffected by the loss of Srap1 (Figure 6E). These observations raise the possibility that additional targeting or repulsion factors might direct the association of Srap1 at the level of individual 5hmC/5fC/5caC-marked CpGs. Interestingly, Srap1 deficiency leads to accumulation of 5hmC/5fC/5caC as well as 5mC, both in ESCs and in subsequent developmental stages. Coupled with reports that Tet1/ or Tet2/ mouse ESCs show a loss of DNA methylation (Hon et al., 2014), these findings implicate the persistence or impaired turnover of 5hmC in the stabilization of neighboring DNA methylated regions. In addition to Srap1, a constellation of functionally uncharacterized proteins, including many with prokaryotic orthologs, have been identified as candidate readers of Tet-oxidized methylcytosine in mammalian cell extracts (Spruijt et al., 2013). Understanding how these proteins function in the regulation of enzymatically oxidized or oxidatively damaged bases, and whether these processes go awry in diseases such as cancer, are goals that await further exploration. EXPERIMENTAL PROCEDURES Srap1 KO Mice and ESCs For gene targeting, a mixture of Cas9 mRNA and single-guide RNA (sgRNA) targeting exon 2 of murine Srap1 was microinjected into the cytoplasm of zygote-stage embryos harvested from 129/Sv females mated with C57BL/ 6N males. Injected embryos were implanted in CD-1 foster mice and pups screened for deletions in Srap1 using PCR primers flanking the targeted region (Table S7). DNA Endonuclease Assays PCR substrates were generated using Phusion Taq polymerase and dATP/ dGTP/dTTP mixed with dCTP, dhmCTP (Zymo Research), dfCTP, or dcaCTP (TriLink Technologies) and purified by spin column. FAM-labeled, HPLC-purified 32-mer DNA oligonucleotides containing 5hmC or 5caC were annealed with complementary oligonucleotide containing 5mC in the bottom strand CpG. Nuclease digestion reactions were performed using PCR-generated substrate (1.5 mg) or labeled oligonucleotide duplex DNA (2.5 mM) mixed with the wild-type or DC2 variants of SRAPd-His6 or GST-Srap1 (0.5 mM) in NEB buffer 2.1 (10 mM Tris-HCl [pH 7.9], 50 mM NaCl, 10 mM MgCl2, and 0.1 mg/mL BSA) in a total volume of 30 mL at 37 C for 1 hr or for the indicated times. For denaturing PAGE, samples were mixed with 17 mL stop buffer (98% formamide, 1 mM EDTA, and 1 mg /mL bromophenol blue) and boiled for 5 min. Products were resolved on 15% TBE-urea gels. For native PAGE, samples were mixed with 6 mL of 63 gel loading dye

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(NEB) and resolved on a 17% acrylamide gel. Gels were imaged using a Gel-Doc XR+ Imager (Bio-Rad). UHPLC-MS/MS Analysis of Genomic DNA Genomic DNA (500 ng) purified from wild-type or Srap1 KO mouse ESCs was treated with DNA Degradase Plus (Zymo Research). Samples (20 mL) were mixed with aqueous formic acid to 0.1% v/v, (final concentration of nucleosides 50 ng/mL) and aliquots injected into a reverse-phase UHPLC column (Eclipse C18 2.1 3 50 mm, 1.8 mm particle size; Agilent) equilibrated with buffer 0.1% formic acid and eluted (200 mL/min) with an increasing concentration of methanol. The areas of each LC-MS measurement were determined by extraction of the accurate parent-daughter ion mass transitions from the total ion current, and the linear fits of the determined area ratios over the known amount gave R2 values of >0.996 for all tested standards. Illumina Library Preparation For ChIP experiments, mouse ESCs stably expressing FHA-Srap1 were fixed in 1% paraformaldehyde for 10 min at 37 C, followed by quenching with 125 mM glycine for 5 min. Cells were lysed and processed using the MAGnify ChIP system (Life Sciences). For DIP, genomic DNA was isolated from Srap1 KO and matched wild-type ESCs using the DNeasy Blood and Tissue DNA kit (QIAGEN) and sonicated for a total of 6 min. Samples were processed using the MeDIP or hMeDIP kits (Active Motif) according to the manufacturer’s protocol. For RNA-seq, total RNA was extracted using the Zymo RNA Clean & Concentrator (Zymo Research), followed by rRNA depletion and cDNA preparation using the KAPA Stranded RNA-Seq Kit with RiboErase. For RRBS, genomic DNA was extracted from E9.5 wild-type and Srap1 KO embryos using the DNeasy Blood and Tissue kit and digested with MspI overnight. Sizeselected (<0.7 kb) DNA fragments were subjected to bisulfite conversion using the EZ DNA Methylation-Gold kit (Zymo Research), followed by 12 cycles of PCR amplification using Kapa HiFi HotStart Uracil+ polymerase. Indexed libraries were prepared using the Kapa HyperPrep kit and purified with 0.93 Ampure XP beads (Beckman Coulter), pooled, and analyzed on an Illumina HiSeq2000 or NextSeq500. Sequencing Data Processing Raw reads were trimmed to remove adapters and low-quality reads using Trim Galore (version 0.3.5). Bowtie (version 0.12.8) was used to obtain reads mapped uniquely to the mouse genome (mm9). ChIP-seq and DIP-seq peak candidates were identified with MACS (version 2.1.1) (Zhang et al., 2008) using input as the control dataset. To remove nonspecific signals, IgG samples were processed similarly and their normalized read density (reads per kilobase of transcript per million mapped reads [RPKM]) values were subtracted from modification-specific peaks. For RNA-seq, Cufflinks (version 2.2.1.0) was used at default settings to assemble transcripts, estimate their abundances, and test for differential transcript expression. For RRBS, trimmed reads were mapped against the reference mouse genome (mm10) with Bismark (version 0.14.5) (Krueger and Andrews, 2011). A chi-square test was used to identify significant DMRs in KO versus wild-type samples, and an FDRadjusted p value < 0.01 was used to determine DMRs. DATA AND SOFTWARE AVAILABILITY The accession number for the RRBS, ChIP-seq, DIP-seq, and RNA-seq data reported in this paper is GEO: GSE81222. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and seven tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2017.09.055. AUTHOR CONTRIBUTIONS D.E.F. conceived the project. S.-M.K. and D.E.F. designed and conducted experiments with cultured cells, performed mouse genetics studies, and

prepared libraries for next-generation sequencing. S.-M.K. and B.Z. conducted biochemical assays. S.-Y.X. and S.-M.K. performed nuclease assays and run-off sequencing. L.A. prepared the scaled sequence LOGO analysis. Y.C., L.A., and D.E.F. analyzed the data. S.-M.K. and D.E.F. wrote the manuscript. ACKNOWLEDGMENTS We thank K. Faull for LC-MS/MS sample processing and A. Rao for helpful discussions. This project is supported by the University of Southern California (USC) Department of Pathology, the Robert E. and May R. Wright Foundation Trust, and the James H. Zumberge Faculty Research and Innovation Fund. Received: March 17, 2017 Revised: August 15, 2017 Accepted: September 18, 2017 Published: October 10, 2017 REFERENCES Amano, T., Hirata, T., Falco, G., Monti, M., Sharova, L.V., Amano, M., Sheer, S., Hoang, H.G., Piao, Y., Stagg, C.A., et al. (2013). Zscan4 restores the developmental potency of embryonic stem cells. Nat. Commun. 4, 1966. Aravind, L., Anand, S., and Iyer, L.M. (2013). Novel autoproteolytic and DNAdamage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems. Biol. Direct 8, 20. Bachman, M., Uribe-Lewis, S., Yang, X., Williams, M., Murrell, A., and Balasubramanian, S. (2014). 5-Hydroxymethylcytosine is a predominantly stable DNA modification. Nat. Chem. 6, 1049–1055. Borgaro, J.G., and Zhu, Z. (2013). Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases. Nucleic Acids Res. 41, 4198– 4206. Dawlaty, M.M., Breiling, A., Le, T., Raddatz, G., Barrasa, M.I., Cheng, A.W., Gao, Q., Powell, B.E., Li, Z., Xu, M., et al. (2013). Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323. Deshpande, R.A., Lee, J.H., Arora, S., and Paull, T.T. (2016). Nbs1 converts the human Mre11/Rad50 nuclease complex into an endo/exonuclease machine specific for protein-DNA adducts. Mol. Cell 64, 593–606.

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