Video imaging of single molecule DNA replication

June 15, 2017 by Andy Fell
Credit: CC0 Public Domain

Almost all life on Earth is based on DNA being copied, or replicated. Now for the first time scientists have been able to watch 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 that raises new questions," said Stephen Kowalczykowski, distinguished professor in microbiology and molecular genetics at the University of California, Davis. The work is published June 15 in the journal Cell with coauthors 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.

Because the two strands in the 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.

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 or off by adding or removing chemical fuel (adenosine triphosphate, ATP) 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.

Stops, Starts and Variable Speeds

Once Graham, Kowalczykowski and Marians started watching individual DNA strands, they noticed something unexpected. Replication stops unpredictably, and when it starts up again can change speed.

"The speed can vary about ten-fold," Kowalczykowski said.

Sometimes the lagging strand synthesis stops, but the leading strand continues to grow. This shows up as a dark area in the glowing strand, because the dye doesn't stick to single-stranded DNA.

"We've shown that there is no coordination between the strands. They are completely autonomous," Kowalczykowski said.

What looks like coordination is actually the outcome of a random process of starting, stopping and variable speeds. Over time, any one strand will move at an average speed; look at a number of strands at the same time, and they will have the same average speed.

Kowalczykowski likened it to traffic on a freeway.

"Sometimes the traffic in the lane one over is moving faster and passing you, and then you pass it. But if you travel far enough you get to the same place at the same time."

The researchers also found a kind of "dead man's handle" or automatic brake on helicase, which unzips DNA ahead of the rest of the enzymes. When polymerase stops, helicase can keep moving, potentially opening up a gap of unwound DNA that could be vulnerable to damage. In fact, exposed single-strand DNA sets off an alarm signal inside the cell that activates repair enzymes.

But it turns out that when it gets uncoupled and starts to run away from the rest of the replication complex, helicase slows down about five-fold. So it can chug along until the rest of the enzymes catch up then speed up again.

This new stochastic approach is a new way of thinking about DNA replication and other biochemical processes, Kowalczykowski said. "It's a real paradigm shift, and undermines a great deal of what's in the textbooks," he said.

Explore further: Single-DNA images give clues to breast cancer

More information: Cell (2017). DOI: 10.1016/j.cell.2017.05.041

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16 comments

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BobSage
1 / 5 (1) Jun 16, 2017
This is beautiful. Reading a description can't compare.

I would like to ask this question: is it truly possible that this mechanism could have evolved? It seems far too complex. I would like to hear someone answer this who does not start with the supposition that evolution is real and then deduce so of course it had to evolve.
RealScience
5 / 5 (1) Jun 16, 2017
is it truly possible that this mechanism could have evolved? It seems far too complex.


Things that evolve get complex. Look at wheeled transportation, for example. From simple log rollers to carts with solid disk wheels, to chariots with rim-and-spoke wheels, to steam locomotives to simple internal combustion engines that you could fix yourself to fancy engines with pollution controls, to electric cars where each wheel is driven by its own motor with rotating magnetic fields, etc.

Or look at the computers we are typing on... and how those have grown from a few thousand vacuum tubes with no operating system and only one application at a time to today's multi-billion transistor multi-core processors with multiple levels of cache memory running the OS, and a browser that lets us see and edit what we are typing...

Is there is anything of comparable complexity whose origins are known for certain that has reached that complexity other than by evolving?
Dingbone
Jun 17, 2017
This comment has been removed by a moderator.
TheGhostofOtto1923
not rated yet Jun 17, 2017
Is there is anything of comparable complexity whose origins are known for certain that has reached that complexity other than by evolving?
Uh none of your examples are evolution. Maybe someone here will want to waste some time trying to explain it to you.
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
Interesting in that it seem to show, in contrast to earlier work, that the replisome is a fully stochastic mechanism with presumably disengaging and re-engaging molecules, as hinted at in the article text. That would be expected on an evolutionary background of polymerase paralogs (from gene duplications) that are engaged in a more complex machinery. It looks like the current mechanism evolved to minimize isolated single strand DNA, which is both engaged by DNA repair mechanisms and contribute to chemical mutability during replication.

As for the experiment, it looks very elegant. I worried that they had used a plasmid rolling circle mechanism, which I do not know much about, but they do let the full replisome assemble in vitro. That is a lot more complex than the video let on, with a "clamp loader" complex that organize the molecules in one huge mechanism. That is by the way presumably the rate organizer, as the helicase is rained in by it. [tbctd]
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
[ctd] If I would criticize the work it is that they show elegant graphs on expected rates - looks Gaussian as expected - and Okazaki fragment primer lengths - look lognormal as expected from random elongation. But they just fit to Gaussians instead of using statistical tests for both the resulting distributions, which would test their model further.
torbjorn_b_g_larsson
5 / 5 (2) Jun 17, 2017
@Bob Sage: I cannot wind history back and pretend that evolution is not an observed fact ,

But if you must I can go back to the discovery, where it is the presence of homologous traits - i.e. similar traits that are inherited, with variation - between species that was the primary evidence and the basis for elucidating the mechanisms of variation and selection for function. It was long noted in extant species, but when it was also noted in the fossil record and its progression, as well as in the biogeography - similar species derive from an ancestral population location - it was the key for the simultaneous Darwin and Wallace discovery (as well as previous but forgotten discoverers some decades earlier).

To wit, the scientific observation is that bird wings evolved from ancestral tetrapod legs, where else would they be inherited from and why else would they have inherited the same bones, muscles and ligaments et cetera? [tbctd]
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
[ctd] Note that the complexity (or the exact putatively complex pathway) is not involved in the test of observing a pathway of an evolutionary process. Complexity has nothing to do with the observation or its scientific test, even if a non-biologist may think so [you can look it up].

I already noted in my first comment that replicases are paralogs, so we can observe straightforward that evolution is the process resulting in the DNA system. I also noted that the observed chemically stochastic independent function of such parts is a more likely result of inheriting gene duplicated molecular copies - later modified under evolution - and other recruited molecular parts that went into the modern system. Finally the Okazaki fragments are a result of DNA replicases having evolved from an ancestral RNA genetic machinery.

A rough estimate is that the time to evolve the DNA genetic machinery from the RNA ancestor was 100 Myrs, about the time taken to evolve land tetrapods from worms.
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
[cont] That estimate is based on the current observation of a habitable ocean before 4.3 billion years ago [Allen et al zircon data, 2015] as well as the many molecular clock datings that agree on that the last universal ancestor of Bacteria and Archaea, which did have the DNA genetic machinery, evolved 4.2 billion years ago. The ocean dating sets a fairly good lower boundary as you can see from the data how the ocean and its habitability gradually grew to a global, habitable one - most likely as emergence habitat - but the molecular clock datings are so-so and it is only their agreement that makes it acceptable as well as the earliest putative fossil candidates now kick in at 4.3 - 4.1 billion years (IMO). YMMV.
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
Let me rephrase that so it is clearer and more informative.

The earliest putative fossil candidates kick in a 4.3 - 4.1 billion years ago. But the earliest putative fossil candidate with putative bacterial affinity - which can possibly be separated between Bacteria and Archaea and be witness to that the last universal common ancestor was older - is > 4.1 billion years ago.

The first result support the ocean habitability date, the latter the Bacteria-Archaea divergence date.
torbjorn_b_g_larsson
5 / 5 (1) Jun 17, 2017
Also, "reined" in.
betterexists
not rated yet Jun 17, 2017
We have 3 Billion Bases in Human Genome; It is Best to Split 26 Bases Each from Einstein's DNA From Beginning To The End & Introduce Them into Zygotes of Different Mice AND SEE WHAT HAPPENS ! Call Them 1-26 Mouse, 27-53 Mouse and So ON if They Do Survive!
betterexists
not rated yet Jun 17, 2017
@BobSage
This is beautiful. Reading a description can't compare.

I would like to ask this question: is it truly possible that this mechanism could have evolved? It seems far too complex. I would like to hear someone answer this who does not start with the supposition that evolution is real and then deduce so of course it had to evolve.

Consider, Unicellular Amoeba and MultiCellular Hydra.
Then Consider Tailed Primates & Tailless Humans.
If you still have Doubt, Go To GOOGLE IMAGES, Search for TAILED HUMANS....For PHOTOS OF Unfortunate Humans Currently Born with Tails!
Our White Blood Cells inside Blood BEHAVE like Amoebae! You may see them in action in Youtube Videos!
RealScience
5 / 5 (1) Jun 18, 2017
... Uh none of your examples are evolution...


None of my examples are of BIOLOGICAL evolution, but they ARE examples of things that have evolved to complexity.

Here are some first definitions of "evolve" in various dictionaries:
Dictionary.com: to develop gradually
Oxford Dictionary: to develop gradually
Cambridge dictionary: to change or develop gradually

Wheeled transport did not jump suddenly from log rollers to a modern car, and computers didn't suddenly jump from a few tubes to multi-core processors with billions of transistors per core and multiple cache hierarchies. They both are the accumulation of countless smaller changes.

Since the question was "is it truly possible that this mechanism could have evolved? It seems far too complex", these are appropriate examples. They are of things evolving to complexity, and they are understandable even by someone skeptical of biological evolution (as BobSage appears to be).
betterexists
not rated yet Jun 18, 2017
We have 3 Billion Bases in Human Genome; It is Best to Split 26 Bases Each from Einstein's DNA From Beginning To The End & Introduce Them into Zygotes of Different Mice AND SEE WHAT HAPPENS ! Call Them 1-26 Mouse, 27-53 Mouse and So ON if They Do Survive!

I think IT IS BEST TO USE VARIOUS BIRD EGGS ! Just Go Down from 26 Bases.
Humans NEED IMMEDIATE RESULTS ! EGGS are Easy To Manipulate. Find Bird Sps. That Hatch Fast! Songbirds take between 10-14 days to hatch and same time to fledge !
betterexists
not rated yet Jun 18, 2017
We have 3 Billion Bases in Human Genome; It is Best to Split 26 Bases Each from Einstein's DNA From Beginning To The End & Introduce Them into Zygotes of Different Mice AND SEE WHAT HAPPENS ! Call Them 1-26 Mouse, 27-53 Mouse and So ON if They Do Survive!

I think IT IS BEST TO USE VARIOUS BIRD EGGS ! Just Go Down from 26 Bases.
Humans NEED IMMEDIATE RESULTS ! EGGS are Easy To Manipulate. Find Bird Sps. That Hatch Fast! Songbirds take between 10-14 days to hatch and same time to fledge !

Small passerines, black-billed and yellow-billed cuckoos

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