Question

You are performing a biochemical purification of enzymes involved in DNA replication. You have purified the replisome. You wish to purify the primosome. You perform further separation techniques on your purified replisome. How will you test to determine that you have purified the primosome

Answer 1

Confirm that the sample has both helicase and primase activities, but not the ability to synthesize DNA

• The replisome is a multiprotein-RNA complex that is required for the replication of DNA. The replisome consists of different proteins which include:

            1. Helicase enzymes that unwind and separate the DNA strand

            2. Replication factor C (RFC), a DNA-dependent ATP that acts as an activator of DNA polymerase.

           3. Proliferating cell nuclear antigen (PCNA)

           4. DNA gyrase/topoisomerase that cuts and reseals DNA strands, which is essential for DNA synthesis.

           5. Primase enzyme that synthesizes short RNA fragments called primers.

          6. DNA polymerase III, an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand.

           7. DNA ligases enzymes that form phosphodiester bonds at a single-strand break in DNA

• A primosome is a multiprotein complex responsible for creating RNA primers on single-stranded DNA (ssDNA) during the replication process.

• The primosome is composed of different primase and helicase enzymes. In Escherichia coli, it consists of PriA helicase, PriB, PriC, DnaB helicase, DnaG (encoded by the dnaG gene), DnaC and DnaT primase.

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