Saturday, May 13, 2017

Hanging On For Dear Life

DNA synthesis on both the leading and lagging strands is done by an integrated, quasi-stable complex.

At the very heart of our hereditary and developmental lives is DNA synthesis- replication that copies one duplex into two. But each beautiful, classic DNA duplex has two strands, each of which needs to be copied. Not only that, but those two strands go in opposite directions. The one is the complement, or anti-sense, of the other one, and in chemical terms heads the other way. This creates a significant problem, since the overall direction of replication can only go one way- the direction of the fork where the parental duplex splits into two.

Complementarity of DNA. When one strand is synthesized in one direction, the other strand has to be synthesized in the opposite direction.

Replication of one of the resulting strands is easy- the "leading strand" is synthesized continuously from 5' starting phosphate onwards as the replication fork progresses. But the other strand, likewise synthesizing from 5' to 3', must head in the opposite direction, away from the replication fork. It is thus called the "lagging strand". Not only must it go in the wrong direction, but it has to work in pieces, synthesizing a short piece away from the replication fork, and then start over again once more single stranded template has been uncovered as the replication fork progresses. These units of newly synthesized DNA on the lagging strand, which are short and separated before they are later repaired and ligated together, are called Okazaki fragments, for the first person to observe and understand them, Reiji Okazaki.

Lagging strand being made in fragments. A helicase unwinding the fork is shown in gold, and the polymerases are show as beige doughnuts. The lagging strand polymerase has to go backwards, away from the direction of fork progression.

Naturally, DNA synthesis happens best when done on a smooth continuous basis, with as few interruptions and errors as possible. So the complexes assembled at the replication fork are quite stable, and held on by special protein clamps which surround the DNA like little doughnuts. An interesting finding was that in addition to the leading strand DNA polymerase, the replication complex also contains the polymerase used for lagging strand DNA synthesis, even while they are heading in opposite directions! This obviously helps keep the whole process orderly, but is a little hard to envision, since in DNA terms, the two polymerases can be hundreds of base pairs apart as they chug along their separate tracks.

More detail on the replication complex. The two polymerases (yellow) doing leading and lagging strand synthesis are actually tethered into one master complex (green), and the lagging strand polymerase, once done with one Okazaki fragment, is yanked back to start the next from near the replication fork. The clamps (red) are loaded by the green central complex in advance of each lagging strand re-initiation.

A recent paper investigated the notorious stability of these complexes. If you dilute an operating replication complex to homeopathic concentrations, the proteins will still stay on track, and continue synthesizing DNA. That indicates that the proteins do not spontaneously fall off by diffusion during their work, or even during the intricate switch between initiation points that is required in lagging strand synthesis. On the other hand, if you add inactive enzyme to ongoing replication complexes, they will grind to a halt, indicating that some kind of polymerase enzyme exchange is going on. What gives?

These authors used a microscopic method and fluorescently labeled polymerases to look at this question in detail. They were able to use a flow cell to stage replication as a visual process, watching a single molecule of DNA extend over time downstream as it was synthesized. With fluorescent polymerase, the progression is even clearer, and a hangup was observed at the first and second Okazaki fragment boundaries (C, the middle lines), since they had not added the ligases that could resolve those boundaries and free up the polymerase to return to the replication fork for another round.

A single DNA molecule being replicated, over time, to longer lengths from a tethered end. When the polymerase is fluorescently labeled, (bottom, purple), it shows the edge of the fork (top) and also the Okazaki fragment boundaries (lower lines).

When a mixture of two different fluorescent polymerases is used, (green and purple), they seem to exchange on the replicating complex, as it goes forth.

But when they used a mixture of two polymerases with different fluorescent colors, the interesting thing was that the colors changed while underway. A fork that started with purple polymerase changed suddenly to green, and then later back to purple. This is very odd for a stable complex. And if they fired a laser at the complex, bleaching its fluorophore permanently, the fluorescence eventually returned, indicating that a new polymerase molecule had hopped on and replaced the original one.

Lastly, if the progressing polymerase is zapped with permanently bleaching light, other fluorescent molecules take its place relatively quickly as the replication proceeds. Bottom is just a graph of the fluorescence intensity from the upper single molecule image.

The replication fork is thus a little like a basketball team. Substitutions can be made while the action is going on, but there can never be less than a full complement of players on the floor. The complexes are stable, but only if there is no other polymerase around. If there are, a new polymerase can jump into the existing fork / complex. It is a clever design that allows resolution of stalled complexes and defunct enzymes, while insuring that the process of replication goes forth as relentlessly and stably as possible.

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