While DNA may not have been the first molecule at the origin of life, it now forms the heart of our replicative and molecular existence, its iconic and elegant stairway storing the digital data that has taken over the world as our wonderful biosphere. Its replication is a complicated process, but has to be done rapidly, since for one of our cells to divide, three billion bases need to be gotten through. A recent paper discussed how that happens, for the DNA polymerase itself can not open up the parental DNA strand fast enough through its own locomotion. It needs a DNA helicase to help unwind the oncoming DNA, whose strands are not only glued together by their basepairs, but can be festooned with histones and all sorts of other bound proteins that need to be cleared away.
To digress slightly, one of the most critical complications in DNA replication is that of the lagging strand. DNA comes in two strands, but all polymerases operate only in one direction (5' to 3'). So replicating the leading strand, which runs 5' to 3', is a piece of cake, even if it requires a helicase for assistance. The other strand is called the lagging strand, and has to be made piecemeal, with the polymerase running "backward" from the replication fork, primed each time by a special RNA-synthesizing priming polymerase. So this process is intrinsically assymetrical and messy.
Anyhow, the researchers use the classic model system of polymerase and helicase from the T7 phage, which infects E. coli bacteria and comes with its own (encoded) stripped-down and efficient DNA replication machinery, for late stages of infection when it needs to pump out full viral genomes at high speed. It is known that the helicase is essential. The polymerase by itself can work, but only slowly, and is particularly retarded by GC-rich regions, the GC nucleotide pair having three hydrogen bonds compared to the AT pair's two bonds. The question for these researchers was.. how closely do the helicase and polymerase work? Are they distant partners, or cheek-by-jowl?
One could imagine that the helicase could proceed some distance ahead of the polymerase. But it is also known that the helicase doesn't work that quickly by itself either. The helicase looks like a lifesaver ring that forms around a single strand of DNA, and consumes dTTP as an energy source to tug on it, pulling it away from the duplex fork which the helicase is trying to melt. Neither activity alone explains the total high rate of DNA replication, suggesting that they work closely to help each other along. The current work put them through their paces either apart or together, and controlled by various concentrations of their inputs, dTTP in the case of the helicase, or all four deoxy-nucleotides in the case of the polymerase.
On a single strand of DNA where it doesn't need to do any helicase-ing, the T7 helicase moves along at a nice 65 nucleotides (nt)/second pace. Likewise, the polymerase, when given plenty of nucleotides and single stranded DNA, chuggs along at 200 nt/second. The final rate of the complex on duplex DNA is about 200 nt/second also, so what needs to be explained is how the polymerase regains that rate when facing duplex DNA and perhaps non-ideal concentrations of reactants, plus other obstacles. For the helicase, how and why does it go faster when yoked to the polymerase than it does on clear single-stranded DNA on its own?
|Performance of DNA polymerase alone, on GC-rich DNA. It is slowed down substantially, but much less when given an excess of its substrates, the deoxynuceotide triphosphates (dNTPs).|
|Key computed parameters of the polymerase alone. With higher GC content in the DNA, the polymerase maximum rate (k-cat) doesn't slow down much, but its responsiveness to substrate concentration( Km) rises substantially.|
|Performance of helicase alone on GC-rich templates. The rate is hugely slowed down no matter what the dTTP (energy substrate) concentration.|
|Unlike the polymerase, the kinetics of the helicase are changed by difficult-to-open GC content mostly in the k-cat, or maximum rate, with little or negative effect in the Km or substrate (dTTP) sensitivity.|
Conversely, the helicase has another problem. No matter how much nucleotide (more precisely, deoxy-nucleotide triphosphate), you give it, it maxes out at pretty slow rates on GC-rich DNA. This suggests that it just isn't a terribly good helicase by itself, but if given a push...
The reason for all this is structural, that the helicase is better at grabbing on to the single strand coming out of the fork, but doesn't have much oomph behind it to plow forward continuously. The polymerase, in contrast, has a good engine, but doesn't grab onto the incoming single strand DNA well, letting it slip back into the duplex with high frequency. The complementary relationship makes sense, since you really do not want the helicase travelling off by itself unwinding the cell's DNA, but rather want it coupled to where it is really needed- right ahead of the DNA polymerase, as a kind of cow-catcher and rail splitter.
|The red nucleotide pictured is fluorescent, and its melting changes the fluorescence signal as measured on the Y-axis of the graphs. Melting in the absence of any dNTP substrate is only substantial when both helicase and polymerase are present.|
Lastly, with some more intricate fluorescence assays, (on which this whole work is based), the authors look at the two or three nucleotides of the fork itself, and which of them are grabbed by the two enzymes in single-stranded form. This is done without giving them any nucleotide triphosphates as energy source or substrate, so it is looking at initial binding. Polymerase or helicase alone bind to the fork, but are pretty ineffective at melting any of the first three nucleotides of the duplex (darker bars). But the two enzymes together melt them quite well (light blue bars). So it is not just the engine of the polymerase behind, but a physically cooperative binding mechanism at the fork that gets the DNA melted in advance of replication.
The authors come up with a highly schematic vision of what this might look like, with some wildly stretched DNA going into the helicase- the green rung of the non-template (right) strand. The helicase then hangs on tightly to each nucleotide that it captures, as the polymerase is busy doing its thing of synthesizing the new DNA strand.
|Model of the polymerase (beige) and helicase (pink) collaborating at the replication fork.|
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