November 2, 2015

First Complete Pictures of Cells' DNA-Copying Machinery

These cartoons show the old "textbook" view of the replisome, left, and the new view, right,
revealed by electron micrograph images in the current study. Prior to this study, scientists
believed the two polymerases (green) were located at the bottom (or back end) of the helicase
(tan), adding complementary DNA strands to the split DNA to produce copies side by side.
The new images reveal that one polymerase is located at the front end of the helicase. The
scientists are conducting additional studies to explore the biological significance of this
unexpected location.

(November 2, 2015)  State-of-the-art electron microscope images reveal that structure of DNA-copying protein complex differs from long-held textbook view

The first-ever images of the protein complex that unwinds, splits, and copies double-stranded DNA reveal something rather different from the standard textbook view. The electron microscope images, created by scientists at the U.S. Department of Energy's Brookhaven National Laboratory with partners from Stony Brook University and Rockefeller University, offer new insight into how this molecular machinery functions, including new possibilities about its role in DNA "quality control" and cell differentiation. The images and implications are described in a paper published online by the journal Nature Structural & Molecular Biology, November 2, 2015.

"This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell," said Huilin Li, a biologist with a joint appointment at Brookhaven Lab and Stony Brook University. "These new images show the fully assembled and fully activated 'helicase' protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two 'polymerase' enzymes that duplicate each strand to copy the genome."

Three-dimensional structure of the active DNA helicase bound to the front-end
DNA polymerase (Pol epsilon). The DNA polymerase epsilon (green) sits
on top rather than the bottom of the helicase.

Studying this molecular machinery, known collectively as a "replisome," and the details of its DNA-copying process can help scientists understand what happens when DNA is miscopied—a major source of mutation that can lead to cancer—or learn more about how a single cell can eventually develop into the many cell types that make up a multicellular organism. But no one has produced a real structure of a replisome at any resolution for any organism until now.

Collaborating scientists and study coauthors Zuanning Yuan, a graduate student
at Stony Brook University (standing), Huilin Li of Stony Brook and Brookhaven Lab
(seated, back), and Jingchuan Sun of Brookhaven Lab (seated, front)
examining protein structures.

"All the textbook drawings and descriptions of how a replisome should look and work are based on biochemical and genetic studies," Li said, likening the situation to the famous parable of the three blind men trying to describe an elephant, each looking at only one part. Those textbook drawings show the helicase moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides (molecules containing the complementary A, T, G, and C bases of the genetic code) to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands.

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