Mre11-Rad50-Xrs2 nicking

DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, Mre11-Rad50-Xrs2 (MRX)-mediated nicking of 5'-terminated DSB ends initiates nucleolytic processing of DSBs and channels repair into the homologous recombination (HR) pathway. How MRX-DNA interactions support 5'-strand specific nicking remains elusive. Here, we monitored millions of MRX nicks at single-nucleotide resolution in vivo. We find that MRX cleaves preferentially 5' of cytosine residues embedded in a sequence with an asymmetric DNA meltability profile. In contrast, nucleosomes and transcription only mildly influence MRX nicking. Our findings suggest that DNA sequence and meltability determine 5'-strand specific MRX cleavage to initiate DSB repair by HR.