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Answer by Surfbanana. Confidence votes 22. 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.
An enzyme called DNA gyrase makes a nick in the double helix and each side separates; An enzyme called helicase unwinds the double-stranded DNA; Several small proteins called single strand binding proteins (SSB) temporarily bind to each side and keep them separated An enzyme complex called DNA polymerase walks down the DNA strands and adds new nucleotides to each strand.