Almost all life on earth is based on DNA being copied, or replicated, and understanding how this process works could lead to a wide range of discoveries in biology and medicine. Now for the first time scientists have been able to watch individual steps in the replication of a single DNA molecule, with some surprising findings. For one thing, there’s a lot more randomness at work than has been thought.

“It’s a different way of thinking about replication that raises new questions,” said Stephen Kowalczykowski, distinguished professor in microbiology and molecular genetics at the University of California, Davis, and at the UC Davis Comprehensive Cancer Center. The work is published June 15 in the journal Cell with co-authors James Graham, postdoctoral researcher at UC Davis, and Kenneth Marians, Sloan Kettering Cancer Center.

Using sophisticated imaging technology and a great deal of patience, the researchers were able to watch DNA from E. coli bacteria as it replicated and measure how fast enzyme machinery worked on the different strands.

DNA replication basics

The DNA double helix is made from two strands that run in opposite directions. Each strand is made of a series of bases, A, T, C and G, that pair up between the strands: A to T and C to G.

The first step in replication is an enzyme called helicase that unwinds and “unzips” the double helix into two single strands. An enzyme called primase attaches a “primer” to each strand that allows replication to start, then another enzyme called DNA polymerase attaches at the primer and moves along the strand adding new “letters” to form a new double helix.

(Look here for a video illustration.)

Because the two strands in the double helix run in opposite directions, the polymerases work differently on the two strands. On one strand — the “leading strand” — the polymerase can move continuously, leaving a trail of new double-stranded DNA behind it.

But on the other, “lagging strand,” the polymerase has to move in starts, attaching, producing a short stretch of double stranded DNA, then dropping off and starting again. Conventional wisdom is that the polymerases on the leading and lagging strands are somehow coordinated so that one does not get ahead of the other. If that did happen, it would create stretches of single-stranded DNA that are highly susceptible to damaging mutations.

The experiment: rolling circles and fluorescent dye

To carry out their experiment, the researchers used a circular piece of DNA, attached to a glass slide by a short tail. As the replication machinery rolls around the circle, the tail gets longer. They could switch replication on by adding chemical fuel (nucleoside triphosphates, NTPs) and used a fluorescent dye that attaches to double-stranded DNA to light up the growing strands. Finally, the whole set up is in a flow chamber, so the DNA strands stretch out like banners in the breeze.