![]() ![]() The two adaptors are identical in sequence but are color coded blue or red to indicate whether the read maps to the top or bottom strand, respectively. Throughout, top strand refers to the strand that runs 5′ to 3′ in the genome assembly. In S1-seq, sequencing adaptors are linked to duplex ends generated by removal of ssDNA tails using nuclease S1. SPO11 (magenta ellipses) cuts DNA via a covalent protein–DNA intermediate. ( B) Early steps in meiotic recombination and overview of the S1-seq method. ![]() ( A) Schematic of H-DNA, consisting of a triplex and ssDNA, formed on a polypyrimidine Spo11-independent S1-seq clusters at H-DNA motifs. This study provides an approach for studying DNA secondary structure genome-wide at high spatial resolution.įig. By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)–specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. Such H-DNA–forming sequences occur frequently in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. polypurine runs, are capable of folding into a triple-helix–containing non–B-form DNA structure called H-DNA. ![]() Certain DNA sequences, including mirror-symmetric polypyrimidine ![]()
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