RecA/Rad51 catalyzed pairing of homologous DNA strands, initiated by polymerization of the recombinase on single-stranded DNA (ssDNA), is a universal feature of homologous recombination (HR). Generation of ssDNA from a double-strand break (DSB) requires nucleolytic degradation of the 5'-terminated strands to generate 3'-ssDNA tails, a process referred to as 5'-3' end resection. The RecBCD helicase-nuclease complex is the main end-processing machine in Gram-negative bacteria. Mre11-Rad50 and Mre11-Rad50-Xrs2/Nbs1 can play a direct role in end resection in archaea and eukaryota, respectively, by removing end-blocking lesions and act indirectly by recruiting the helicases and nucleases responsible for extensive resection. In eukaryotic cells, the initiation of end resection has emerged as a critical regulatory step to differentiate between homology-dependent and end-joining repair of DSBs.