DNA Copier Component Found to Be Real Drag

Enzyme that Builds Primer on Lagging Strand Acts as Brake on Leading Strand

A new study sheds light on a longstanding puzzle in DNA replication: how do the enzymes that copy the two strands of DNA manage to coordinate their separate movements while remaining in sync as part of a single replication complex? The answer, detailed in the Feb. 2 Nature by Antoine van Oijen’s lab, is that replication on the leading strand, which can progress much more quickly, pauses to wait for replication on the lagging strand to catch up. The enzyme that constructs short primers on the lagging strand acts as a brake to slow the leading strand’s advance.

Photo by Graham Ramsay

Antoine van Oijen (center), Jong-Bong Lee (right), and Samir Hamdan found that in the dance between partner DNA polymerases during replication, the leading strand waits a beat for the lagging strand to catch up.

Catching this little pas de deux in action required watching the process at a single-molecule level. By taking advantage of the mechanical properties of DNA, the team was able to track the pace of these reactions in real time.

Replicating the genome is complicated by a quirk of DNA chemistry: DNA has polarity, and the two strands run like a two-lane highway in opposite directions. But DNA polymerase can only copy DNA in one direction. The two DNA polymerases are part of a single complex, chugging along the two strands as if on a one-way road. The polymerase on the leading strand is moving with traffic, so it can forge ahead without stopping; the polymerase on the lagging strand must copy the DNA backwards in fragments.

This model has been known for decades, but a few questions have remained. For one, if the two DNA polymerases are yoked together into one complex, how do they coordinate their very different steps? DNA polymerase can speed along at 500 base pairs of DNA per second. But each new fragment on the lagging strand requires the construction of a short RNA primer, a process that takes about a second. Van Oijen, HMS assistant professor of biological chemistry and molecular pharmacology, wanted to see why these frequent stops do not result in the lagging strand getting hopelessly behind the leading strand.

To look at how the timing of the two strands is coordinated, van Oijen and his team, including first author and research fellow Jong-Bong Lee, came up with an approach that allows them to “actually see what happens in real time on a single DNA molecule,” he said. For simplicity, they used the replication machinery of the bacteriophage T7, which contains a pared-down, four-protein version of the larger replication complex that higher organisms have. Lee and research fellow Samir Hamdan worked with Charles Richardson, the Edward S. Wood professor of biological chemistry and molecular pharmacology, who has studied the T7 system for decades. The team took a stretch of DNA and anchored one end of the lagging strand to the inner surface of a glass flow cell; on the other end, they affixed a latex bead. When they allowed fluid to flow across the DNA, the bead would drag the DNA in the direction of the current, stretching it out slightly. Under a constant flow rate, the DNA would remain at a constant length.

With the DNA anchored like this, the team could take advantage of the special mechanics of the molecule. “Double-stranded DNA shows a completely different behavior than single-stranded DNA,” van Oijen said. When some of the DNA becomes single stranded, it coils up, making the length of the entire strand shorter. By training a simple optical microscope with a camera on an entire field of beads linked to DNA, the researchers were able to use the position of the beads as an indicator of how much DNA was single- versus double-stranded. By tracking the lagging strands as they lengthened and shortened, the researchers could piece together the events taking place on both strands.

As a first experiment, the team initiated replication, but allowed DNA polymerase to attach only to the leading strand. They were able to see the lagging strand become progressively shorter as the leading strand was copied, leaving the lagging strand to coil up into single-stranded DNA.

When both the strands were replicated, the picture was more complex, and it required some mathematics to extrapolate events on each strand from the bead’s movement. In this case, the polymerase on the leading strand did not simply chug along at a constant pace; the researchers were able to see small pauses in the leading strand’s synthesis when a primer was formed on the slower strand. “It stops to allow the slow process of making this primer to take place,” said van Oijen.

When the researchers interfered with the function of the primase that constructs RNA primers, the pauses disappeared. The pauses also stopped when the ribonucleotides needed to construct primers were removed. The timing of the pauses also corresponded well with known primase binding sites in the lagging strand. This suggests that while the primase constructs a primer on the lagging strand, it also acts as a brake keeping the polymerase on the leading strand from zooming ahead, though the exact braking mechanism is still unclear.

Replication dynamics. Changes in the length of a DNA molecule during replication (a) reveal a pause when the primer is synthesized on the lagging strand (I). The DNA then gets shorter as replication continues on both strands (II), followed by a jump in length when loops formed by the lagging strand are released (III). Figure b shows a schematic of the same events.

The team also saw further proof of one aspect of the current model of DNA replication. As each fragment grows on the lagging strand, the copied DNA is thought to form a loop like a belt pulled through a buckle, which is then released; this allows the polymerase to stay physically linked to the cell’s replication machinery even as it copies in reverse. Van Oijen’s team was able to see shifts in the DNA length that corresponded to the release of these loops.

Van Oijen said that these subtle movements could not be seen by conventional biochemical assays. He compares it to a race in which a single hurdle is placed in each lane of the track; knowing the average position of the runners at any one time would never reveal the pause that each runner takes to jump over the hurdle. “It’s a short time period, and different points of replication will be out of phase with others,” he said. Single-molecule studies like these are attempting to bridge the gap between biochemistry and structural biology, which can show highly detailed snapshots of molecules, but only outside of their biological context.

Taekjip Ha, a Howard Hughes investigator at the University of Illinois at Urbana–Champaign, who works on similar techniques, said that the study “gives a very simple solution to a longstanding problem. It could go into a textbook.” The technique that van Oijen’s team used is much simpler than some of the more high-tech single-molecule approaches like optical tweezers. “The tools are simpler,” Ha said, “but because of their ease of use, [the researchers] could actually study more complex systems.” Previous studies on DNA replication have focused on the DNA helicase, polymerase, or primase in isolation; this is the first study to tackle all three activities at once.

—Courtney Humphries

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