The DNA of an organism suffers from external environmental stresses as well as internal insults and errors resulting from replication and recombination. There are multiple DNA repair pathways to maintain the integrity of the genome. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. This chapter summarizes the role of nucleases in DNA repair, replication and recombination. One of such nucleases, flap endonuclease-1 (FEN-1), is discussed in detail as a multifunctional and structure-specific nuclease involved in several nucleic acid processing pathways, including RNA primer removal, long patch base excision repair, and the resolution of di- and tri-nucleotide repeat secondary structures and stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including the formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage, and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington’s disease, myotonic dystrophy and cancers.