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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

In the Pipeline

« Product Inhibition, Or Grinding To A Halt | Main | The Supreme Court Rules on Myriad »

June 13, 2013

Watching DNA Polymerase Do Its Thing

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Posted by Derek

Single-molecule techniques are really the way to go if you're trying to understand many types of biomolecules. But they're really difficult to realize in practice (a complaint that should be kept in context, given that many of these experiments would have sounded like science fiction not all that long ago). Here's an example of just that sort of thing: watching DNA polymerase actually, well, polymerizing DNA, one base at a time.

The authors, a mixed chemistry/physics team at UC Irvine, managed to attach the business end (the Klenow fragment) of DNA Polymerase I to a carbon nanotube (a mutated Cys residue and a maleimide on the nanotube did the trick). This give you the chance to use the carbon nanotube as a field effect transistor, with changes in the conformation of the attached protein changing the observed current. It's stuff like this, I should add, that brings home to me the fact that it really is 2013, the relative scarcity of flying cars notwithstanding.

The authors had previously used this method to study attached lysozyme molecules (PDF, free author reprint access). That second link is a good example of the sort of careful brush-clearing work that has to be done with a new system like this: how much does altering that single amino acid change the structure and function of the enzyme you're studying? How do you pick which one to mutate? Does being up against the side of a carbon nanotube change things, and how much? It's potentially a real advantage that this technique doesn't require a big fluorescent label stuck to anything, but you have to make sure that attaching your test molecule to a carbon nanotube isn't even worse.
KF%20graphic.jpg
It turns out, reasonably enough, that picking the site of attachment is very important. You want something that'll respond conformationally to the actions of the enzyme, moving charged residues around close to the nanotube, but (at the same time) it can't be so crucial and wide-ranging that the activity of the system gets killed off by having these things so close, either. In the DNA polymerase study, the enzyme was about 33% less active than wild type.

And the authors do see current variations that correlate with what should be opening and closing of the enzyme as it adds nucleotides to the growing chain. Comparing the length of the generated DNA with the FET current, it appears that the enzyme incorporates a new base at least 99.8% of the time it tries to, and the mean time for this to happen is about 0.3 milliseconds. Interestingly, A-T pair formation takes a consistently longer time than C-G does, with the rate-limiting step occurring during the open conformation of the enzyme in each case.

I look forward to more applications of this idea. There's a lot about enzymes that we don't know, and these sorts of experiments are the only way we're going to find out. At present, this technique looks to be a lot of work, but you can see it firming up before your eyes. It would be quite interesting to pick an enzyme that has several classes of inhibitor and watch what happens on this scale.

It's too bad that Arthur Kornberg, the discoverer of DNA Pol I, didn't quite live to see such an interrogation of the enzyme; he would have enjoyed it very much, I think. As an aside, that last link, with its quotes from the reviewers of the original manuscript, will cheer up anyone who's recently had what they thought was a good paper rejected by some journal. Kornberg's two papers only barely made it into JBC, but one year after a referee said "It is very doubtful that the authors are entitled to speak of the enzymatic synthesis of DNA", Kornberg was awarded the Nobel for just that.

Comments (5) + TrackBacks (0) | Category: Analytical Chemistry | Biological News | The Scientific Literature


COMMENTS

1. Keith Robison on June 13, 2013 9:27 AM writes...

It's worth noting that the Pacific Biosciences Single Molecule Real Time (SMRT) DNA sequencer watches tens of thousands of polymerases simultaneously in real time. They've already done neat work showing how modified bases alter the kinetics of incorporation, and I suspect a really clever person could generate mutant polymerases linked to DNA barcodes and explore very large mutational landscapes.

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2. DCRogers on June 13, 2013 9:45 AM writes...

Breaking news: US Supreme Court rules genes cannot be patented.

In associated I-don't-get-this news, Myriad Genetics stock price up. Must be a story here, or a subtlety of the ruling, that I'm not getting.

Permalink to Comment

3. daen on June 13, 2013 1:34 PM writes...

@2: The markets had discounted Myriad to reflect the uncertainty of the SC ruling against them on all claims. That they didn't restored the price to what must presumably be closer to the fair value.

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4. newnickname on June 14, 2013 8:00 AM writes...

(I submitted a comment yesterday, it was posted, and now it's gone. What's up with that? Basically, I said ...)

As I recall, the original Kornberg papers describe more of an ACTIVITY than a purified protein or enzyme. By today's or even 1990's standards, it was slop. On the other hand, with the tools of the day (1957), you must give credit (Nobel Prize) where credit is due. However, I can see a nitpicking referee questioning what they actually had in their stuff that had polymerase activity and any claim of a specific substance.

I also noted that publication was facilitated by the JBC Editor-in-chief who overruled the refs. Not everyone benefits from such intervention. Pipeline, Retraction Watch and others frequently note papers that should never have been published in the first place. No one is able to track rejected, unpublished mss whose results are duplicated or validated by others many years later, without any sort of recognition or awareness at all. The fraudulent research gets more recognition than good science blocked from publication! (Reminds me of politicians: "Bad news is better than no news."

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5. Gregory Weiss on June 19, 2013 4:20 PM writes...

After many years of reading, always enjoying, and very occasionally disagreeing with Derek Lowe's "In the Pipeline," it's a real thrill to see my own lab's work featured here. Thanks a lot for the accurate summary and recognition. Your attention to detail is impressive.

I met Arthur Kornberg when I was a postdoc at Genentech (he visited to present a seminar on polyPi). He had truly infectious enthusiasm, and asked everyone meeting him to sign his notebook. This habit turned celebrity on its head. "Hey, the Nobelist wants my signature!?!" I also wish I had the opportunity to talk to him about these experiments. I would love to see his reaction...

Thanks again for this post and all of the insights over the years.
-Greg

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