A. DNA polymerase adds deoxyribonucleotides to the 3'-OH end of the growing polynucleotide chain.
B. Helicase helps in unwinding the two strands of DNA double helix during replication.
C. Ligase joins the DNA strand by catalyzing the formation of a phosphodiester bond between the adjacent bases.
D. Topoisomerase helps in relieving the tension created during the unwinding of DNA.
Hence, the enzyme that catalyzes the separation of DNA strands during DNA replication is Helicase.
So, the correct answer is 'Helicase'.