Optimized Methods for Mapping DNA Double-strand-break Ends and Resection Tracts and Application to Meiotic Recombination in Mouse Spermatocytes

Overview

Journal bioRxiv

Date 2024 Aug 16

PMID 39149289

Authors

Soonjoung Kim

Shintaro Yamada

Kaku Maekawa

Scott Keeney

Affiliations

Soon will be listed here.

Abstract

DNA double-strand breaks (DSBs) made by SPO11 protein initiate homologous recombination during meiosis. Subsequent to DNA strand breakage, endo- and exo-nucleases process the DNA ends to resect the strands whose 5´ termini are at the DSB, generating long 3´-terminal single-stranded tails that serve as substrates for strand exchange proteins. DSB resection is essential for meiotic recombination, but a detailed understanding of its molecular mechanism is currently lacking. Genomic approaches to mapping DSBs and resection endpoints, e.g., S1-sequencing (S1-seq) and similar methods, play a critical role in studies of meiotic DSB processing. In these methods, nuclease S1 or other enzymes that specifically degrade ssDNA are used to trim resected DSBs, allowing capture and sequencing of the ends of resection tracts. Here, we present optimization of S1-seq that improves its signal:noise ratio and allows its application to analysis of spermatocyte meiosis in adult mice. Furthermore, quantitative features of meiotic resection are evaluated for reproducibility, and we suggest approaches for analysis and interpretation of S1-seq data. We also compare S1-seq to variants that use exonuclease T and/or exonuclease VII from instead of nuclease S1. Detailed step-by-step protocols and suggestions for troubleshooting are provided.

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