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Novel Approach Reveals Genomic Landscapes of Single-Strand DNA Breaks with Nucleotide Resolution in Human Cells

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Novel Approach Reveals Genomic Landscapes of Single-Strand DNA Breaks with Nucleotide Resolution in Human Cells ARTICLE https://doi.org/10.1038/s41467-019-13602-7 OPEN Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells Huifen Cao1,5, Lorena Salazar-García 1,5, Fan Gao1, Thor Wahlestedt1, Chun-Lin Wu2, Xueer Han1, Ye Cai1, Dongyang Xu1, Fang Wang1, Lu Tang1, Natalie Ricciardi3, DingDing Cai1, Huifang Wang1, Mario P.S. Chin 1, James A. Timmons4, Claes Wahlestedt 3* & Philipp Kapranov1* 1234567890():,; Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely con- tribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches. 1 Institute of Genomics, School of Biomedical Sciences, Huaqiao University, 668 Jimei Road, Xiamen 361021, China. 2 Department of Pathology, Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, China. 3 Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA. 4 Augur Precision Medicine LTD, Scion House, Stirling University Innovation Park, Stirling FK9 4NF, UK. 5These authors contributed equally: Huifen Cao, Lorena Salazar-García. *email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2019) 10:5799 | https://doi.org/10.1038/s41467-019-13602-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13602-7 NA damage is now widely recognized as a major reason purification and reduces its size to a range suitable for down- Dbehind cancer and many other aging-associated diseases stream NGS analyses. Prior to fragmentation, cells are crosslinked and as such represents a very important issue for human in situ with formaldehyde. Nuclei isolated from the crosslinked health1,2. While multiple types of DNA lesions exist, SSBs are cells are then treated with MNase to fragment genomic DNA to a considered the most common type of DNA damage3. These range of 150–500 base pairs (Fig. 2a). Following MNase inacti- lesions can represent sites of oxygen radical DNA damage, vation, the nuclei are subjected to proteinase K treatment and intermediates in excision DNA repair pathway and products of crosslink reversal. The fragmented genomic DNA is then isolated, unresolved intermediates of enzymes such as topoisomerases3. denatured and polyA-tailed with TdT. The polyA tags can then be SSBs can further deteriorate into highly toxic double-strand used to capture and identify the positions of SSBs genome-wide breaks (DSBs) by stalling or collapsing replication forks4. How- using NGS (Fig. 1). ever, by themselves SSBs can also represent a major issue for cells Initially, we tested utility of this method using a 3rd generation as they can inhibit progression of RNA polymerase5 and in some NGS platform based on Helicos SMS technology15 ideally suited cases cause apoptosis6,7. The importance of this type of lesion is for this task. This platform can directly sequence single-stranded underscored by existence of dedicated cellular pathways that deal (ss)DNA16 possessing 3′ polyA tails on a flow-cell containing with every step of fixing SSBs from detection to processing to millions of oligo(dT) molecules16 that can both capture the repair3. Defects in these pathways can lead to cellular sensitivity polyA-tailed molecules and prime sequencing reactions (Fig. 1). to genotoxic stress, embryonic lethality and a number of neuro- Thus, the tailed native genomic DNA can be directly used for degenerative diseases8. SMS without any additional amplification or preparation steps The remarkable progress in appreciation of the fine details of (Fig. 1). We applied this strategy (named SSiNGLe-SMS) to the SSB repair machinery, however, stands in stark contrast with profile SSBs in human leukemia K562 cells treated with different total absence of methods to map endogenous SSBs in a global, anti-cancer medications (see Methods) and PBMC’s isolated from unbiased and genome-wide fashion with nucleotide precision. 40 donors. After filtering aligned reads for proximity to internal This gap in available methodologies also contrasts with a suite of polyA stretches in the genome, 2–4% of reads remained in the comprehensive approaches developed for mapping DSBs with un-tailed controls compared to 20–60+% in the tailed samples nucleotide-level resolution, such as BLESS9, BLISS10 and others with median 42.6% in both sample types (Fig. 2b, Supplementary (reviewed in ref. 11). To the best of our knowledge, only one SSB Data 1). Furthermore, the number of the post-filtered reads in the genome-wide mapping method can provide comprehensive and untailed controls corresponded to only 0.2–3.4% of median unbiased data12. The procedure relies on the 3′OH group of an number of post-filtered reads in the tailed samples (Fig. 2b, SSB to prime a DNA polymerase I nick-translation reaction that Supplementary Data 1). labels downstream DNA with a biotinylated nucleotide12. The Encouraged by these results, we further sought to adapt this labeled DNA is then purified and subjected to next-generation approach to a more widely used Illumina NGS platform that also sequencing (NGS)12. However, this approach maps a region of generates longer reads allowing for a more accurate mapping of DNA, quite possibly extending thousands of bases from the ori- SSBs. To accomplish this, we added the following three key steps to ginal SSB, thus precluding identification of the lesion with the SMS protocol (Fig. 1). First, the polyA-tailed molecules were nucleotide precision. On the other hand, a nucleotide-level linearly amplified using 10 cycles of primer extension with a 13 – method to map sites of excision repair has been developed ; chimeric DNA RNA oligo-d(T)50-r(T)3 oligonucleotide consisting however, it cannot provide information on breaks generated by of 50 2′-deoxythymidine and 3 thymidine nucleotides at the 3′ end. other mechanisms. The last 3 RNA residues were included to prevent tailing of the Thus, all this leads to a total dearth of knowledge of nucleotide- oligonucleotide with TdT at the next step since the enzyme cannot level genome-wide patterns for this critical type of DNA lesion. use RNA as a substrate. Second, the products of primer extension Here, we develop and validate an approach, SSiNGLe (single- were tailed at the 3′ end with polyC tail using TdT. Finally, the strand break mapping at nucleotide genome level) that can pro- desired molecules containing oligo-d(T)50-r(T)3 sequences at the 5′ vide nucleotide-level maps of native SSBs genome-wide. We ends and polyC-tail at the 3′ ends were exponentially PCR implement this approach to work with two NGS platforms: (1) a amplified with oligonucleotides containing Illumina adaptor 3rd generation single molecule sequencing (SMS) platform sequences (Fig. 1) and subjected to NGS. As in the SMS (Helicos/SeqLL) whose unique capabilities obviate the need for experiments, samples without the polyA-tailing served as controls. lengthy sample preparation and PCR amplification, and (2) the We then further tested various performance metrics of the more commonly used Illumina platform. We show highly con- Illumina based approach (SSiNGLe-ILM). First, we tested sistent and striking patterns of SSBs using both sequencing whether it could indeed detect SSBs by using a nicking site- platforms. Furthermore, the results show that the genomic pat- specific restriction endonuclease Nt.BbvCI that can digest only tern of breaks—the SSB “breakome”—has strong potential to the top strand of CCTCAGC site. Crosslinked nuclei were represent a novel dimension describing state of a biological sys- digested with Nt.BbvCI prior to MNase fragmentation. Indeed, as tem and a novel source of blood-based biomarkers, with poten- shown in Fig. 2c, we observed a very high enrichment (152-fold) tially yet undiscovered connections to aging. of breaks mapping to the correct strand of the CCTCAGC site. Second, digestion with this enzyme allowed to test whether the method indeed has nucleotide-level precision. In fact, about half Results (43.3–50.7%) of the breaks mapping within ±5 bp of Nt.BbvCI cut Overview and validation of SSiNGLe-SMS and SSiNGLe-ILM. sites mapped to the correct base and the vast majority The essence of our approach is based on tagging a free 3′-OH (86.8–94.7%) were within 1 nucleotide of the predicted cut site terminus representing an SSB by addition of the polyA tail using (Fig. 2c, Supplementary Data 2). Third, we tested whether breaks terminal transferase (TdT) (Fig. 1). Prior to the tagging, the high- with termini other than 3′-OH could be detected. To accomplish molecular weight (HMW)
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