Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other

Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base pair partially. When combined, the junction base pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA. DOI: http://dx.doi.org/10.7554/eLife.06562.001
eLife digest DNA replication is the process whereby a molecule of DNA is copied to form two identical molecules. First, an enzyme called a DNA helicase separates the two strands of the DNA double helix. This forms a structure called a replication fork that has two exposed single strands. Other enzymes called DNA polymerases then use each strand as a template to build a new matching DNA strand. DNA polymerases build the new DNA strands by joining together smaller molecules called nucleotides. One of the new DNA strands—called the ‘leading strand’—is built continuously, while the other—the ‘lagging strand’—is made as a series of short fragments that are later joined together. Building the leading strand requires the helicase and DNA polymerase to work closely together. However, it was not clear how these two enzymes coordinate their activity. Now, Nandakumar et al. have studied the helicase and DNA polymerase from a virus that infects bacteria and have pinpointed the exact positions of the enzymes at a replication fork. The experiments revealed that both the polymerase and helicase contribute to the separating of the DNA strands, and that this process is most efficient when the helicase is only a single nucleotide ahead of the polymerase. Further experiments showed that the helicase stimulates the polymerase by helping it to bind to nucleotides, and that the polymerase stimulates the helicase by helping it to separate the DNA strands at a faster rate. The next challenge is to investigate the molecular setup that allows the helicase and polymerase to increase each other's activities. DOI: http://dx.doi.org/10.7554/eLife.06562.002