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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.
These replication forks are the actual site of DNA copying. Helix destabilizing proteins bind to the single-stranded regions so the two strands do not rejoin. Enzymes called topoisimerases produce breaks in the DNA and then rejoin them in order to relieve the stress in the helical molecule during replication. As the strands continue to unwind and separate in both directions around the entire DNA molecule, the hydrogen bonding of free DNA nucleotides with those on each parent strand produces new complementary strands.