The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.
Before a cell divides, it must duplicate its DNA so that each new cell inherits its own copy of the genome. To do this, the DNA double helix must be unwound so that the two individual strands of DNA can serve as templates for making new DNA molecules. Unwinding begins when two helicase complexes, termed the Mcm2-7 rings, are loaded together onto the DNA.
At first, the two Mcm2-7 rings encircle the double-stranded DNA and remain bound together in an inactive form. Activating the Mcm2-7 rings requires the binding of five other proteins to each ring, which forms two larger complexes called CMG helicases. When the CMG helicases form, the two DNA strands separate and an individual Mcm2-7 ring ends up encircling each of the single DNA strands. However, how an activated CMG complex is assembled, and how it binds to and unwinds DNA, is not fully understood.
Now, Costa et al. have determined the three-dimensional structure of the fruit fly CMG helicase bound to a DNA double helix with a single-stranded overhang at one end. The activated Mcm2-7 ring binds to the overhang, which confirms previous findings indicating that the activated helicase prefers single-stranded over double-stranded DNA. The structure also shows that, as a CMG helicase slides along the single-stranded DNA towards the double-stranded DNA, it is the ring complex's ‘motor domains’ that lead the way, while its DNA-binding domains trail behind.
Costa et al. also found that disrupting some of the interactions between two of the five proteins that bind to the Mcm2-7 ring either prevented the replicative helicase from forming or made it unstable. Furthermore, it was revealed that one of these two proteins—called Cdc45—was ideally placed to capture the strand of DNA that might be accidentally released from the Mcm2-7 ring. It was also discovered that when the complex is bound to DNA, the motor domains of the Mcm2-7 complex change shape from a flat ring to a spiral structure; the DNA-binding domains, however, remain in a flat ring. Costa et al. note that this structure is similar to that adopted by many viral and bacterial helicases, and that it even shares many features with the molecular machinery that breaks down unneeded or damaged proteins inside cells.
Finally, Costa et al. were able to image a structure composed of two CMG complexes bound together. This reveals the relative orientation of the two Mcm2-7 rings before they separate and move in opposite directions to unravel the DNA. The findings of Costa et al., combined with previous structural work in this field, demonstrate that the Mcm2-7 helicase complex can adopt many different shapes as it is assembled on DNA and activated to support DNA replication.