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An exonuclease is an enzyme that catalyse the hydrolysis of single nucleotides from the end of a DNA or RNA chain, by breaking the phosphodiester bonds. The nucleotides are removed one by one. They are used for metabolism and maintaining the genome. Exonucleases can act as proof-readers during DNA polymerisation in DNA replication, to remove unusual DNA structures that arise from problems with DNA replication fork progression, and they can be directly involved in repairing damaged DNA.

They are different to endonucleases as the endonucleases hydrolyse phosphodiester bonds at the middle of the DNA chain.

They are useful for RNA degradation and can work in the 5' to 3' or 3' to 5' direction depending on the exonuclease.

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