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Helicase and RNA polymerase separate DNA strands by breaking the hydrogen bonds between complementary bases.

Helicase parts the strands of DNA during DNA replication, and RNA polymerase parts them during transcription.
The enzyme that separates DNA in called DNA helicases. There are two of them that work away from the origin of replication, creating in "bubble" in the DNA molecule. For eukaryotes, there would be several origins of replication but in prokaryotes, there is only one origin of replication.

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