Recombinant DNA and Biotechnology

Enzymes that travel along DNA to copy or transcribe it — the crucial processes underlying cell replication and protein production — aren’t coordinated by a central dispatcher. In fact, they often collide. Now, Rockefeller University researchers have discovered that when DNA-copying machines run head-on into proteins performing less critical tasks, they kick the obstacles aside and continue on their way. The finding, reported in the January 29 issue of Science, reveals new details about the “rules of the road” that help cells make accurate copies of their genetic material — essential for producing healthy offspring.

In preparation for cell division, cells rely on complex protein machines called replisomes to untwist and tease apart the double helix of DNA. As the two strands separate, the replisome copies the strands, producing two complete sets of the genome. The replisome moves at high speed for long distances on DNA, but it runs along the same path as the RNA polymerases that transcribe DNA into messenger RNA, the genes’ instructions for manufacturing proteins. Sometimes these convoys move in opposite directions and collisions are unavoidable.

To find out what happens when they collide, Michael O’Donnell, head of Rockefeller’s Laboratory of DNA Replication and a Howard Hughes Medical Institute investigator, and his colleague Richard Pomerantz reconstructed a cellular traffic accident in a test tube. They developed a system that allowed them to assemble the replisome from the relatively simple bacteria Escherichia coli at one end of a DNA strand — a years-long endeavor in O’Donnell’s lab — and then set it on a collision course with a stalled RNA polymerase from the opposite direction. The scientists found that the DNA replication machine managed to copy the full length of the DNA molecule, indicating that it had traveled the entire distance, despite the obstacle. Further analysis of the collision suggested that the replisome stops when it encounters the RNA polymerase, shoves the RNA polymerase off the DNA and then proceeds.