Saturday, August 27, 2011

Breaking DNA to save itself

The structure of an enzyme that disentangles DNA- by passing strands through each other.

A single break in a cell's DNA is, typically, lethal. The cell will wait and wait for repair to happen, but if it doesn't, boom- it commits suicide. This is one of the quality controls that cancerous cells lose, in order to carry on despite the broken chromosomes they typically contain, among many other mutations. It is one of many homeostatic and quality control mechanisms that manage our cells. Another enforces that all DNA is completely replicated before cell division begins, and a different one enforces that all the condensed chromosomes are congregated neatly at the middle of the cell in mitosis, before separation and division can proceeed.

Yet we have enormous amounts- eight feet- of DNA in each cell, wrapped up in knarly bundles that can't possibly be maintained tangle-free, even with nice rollers to curl on (histones) and scaffolds to fold into (chromosomes, when condensed). On top of that, DNA is helical and additionally twisted, requiring unwinding to be read by RNA polymerases, and much more extensive unwinding to be replicated. What we find inside our cell nuclei is a mess. So naturally, we have several enzymes dedicated to untangling DNA- winding and unwinding it, and in extremis, when a knot can't be unwound, an enzyme that passes strands through each other, cutting the gordian knot.

These are topological problems, so the enzymes are called topoisomerases, catalyzing transitions between topological states. The current paper describes an atomic structure of topoisomerase II, which cuts DNA, allows another strand to pass through the cut, and then reseals the original strand. Quite a dangerous proposition! The experimenters used an anti-cancer drug (etoposide) to lock the enzyme in an interesting halfway state of cut DNA, helping them grow the crystals of protein that provided the structure.

Remember that the diffraction pattern of X-rays passed through a crystal allows mathematical reconstruction of the arrangement of the crystal's atoms, given enough order in that crystal, and enough intensity from the X-rays, typically provided by synchrotrons these days.

X-ray diffraction pattern of an arbitrary crystal. The center is where the main X-ray beam goes through, and the surrounding dots are reflections from the atomic crystal planes. With a lot of math, one can reconstruct the crystal's atomic structure.
In cancer cells, the DNA is particularly messed-up, cell devision is rapid, and the quality control mechanisms that tell the cell to halt and wait for repairs when the DNA is broken are gone. So this drug encourages more and more DNA breaks, to the point that active cancer cells get fatally damaged, even without the specific suicide system that is sensitive to single DNA breaks in normal cells. The cancer cells are given the rope to hang themselves.

But I am more interested in the magic of the topoisomerase II enzyme. (Topoisomerase I enzymes just nick one strand of the DNA, altering its helical winding- a much less complex proposition). It is interesting to consider how mere enzymes could effectively untangle DNA as they do. They don't have fingers or eyes, and they don't have any wider perspective on what is going on in the cell or in the DNA knots that evolution has fashioned them to resolve. They just cut DNA and reseal it, but in a clever way that leads, quite efficiently, to de-knotting of the cell's DNA.

The cycle of action is shown below, in cartoon form.

The enzyme grabs one segment of DNA (the G-segment), and bends it. This bend plays a critical role in funneling local knotted DNA segments (the T-segment in this case) which topologically "want" to pass through the G-strand towards the "mouth" of the enzyme, here shown in beige. When such a T-segment arrives, the enzyme, using ATP, cleaves the G-segment, opens its DNA gate (red, and see below), and allows the T-strand to pass through to the C-gate, the hollow area below the active site (green). Lastly, the G-strand is resealed, the T-segment is released, and everything is reset as it was at the start, minus one tangle. Incidentally, bent, stressed DNA induces increased activity by this enzyme.

Why does the C-gate exist? One might think that once the T-strand is through, then no problems- no need to keep it around rather then let it go on its way. I think the reason is for informational control- so that the enzyme knows to reseal the G-strand, rather than to cut it again. I assume there is a shape-dependent control by the occupied C-gate to enforce the direction of the overall cycle of the cutting/sealing active site.

The structure of the enzyme (just the core part, including only the colored areas of the protein cartoon in "A") is shown below. The C-gate is hard to miss. This large void is clearly able to hold the passed T-strand of DNA while the enzyme ligates the G-strand back to its pristine condition. Looking carefully, one can also see the strong bend of the G-segment DNA (backbone in blue), with both ends pointing sharply upwards.

One can imagine that the rest of the enzyme that was not solved or shown here (gray in part A) might help to form more of the funnel that brings the T-segment into proper position at the top. It might also help the enzyme hold tighly onto those DNA ends that, were they to get lost, would be virtually impossible to find again in the vast molecular soup of the cell and likely cause complete cellular arrest and death.

The cancer drug and topoisomerase II inhibitor, etoposide (in yellow) blocking the DNA strands within the topoisomerase II complex from being fully religated.

The paper devotes most of its time to the structure of the etoposide drug complex- how it locks the enzyme in an intermediate conformation and how these interactions might guide the design of better drugs. Given that this whole mode of therapy is rather crude, (hardly better than bombarding cells with radiation), it is hard to imagine how any "improvements" to the inhibitor would be helpful. Nevertheless, I find these structures immensely interesting- informative about how our bodies work at the molecular level, enlightening about obvious questions that arise with the advent of ever-longer DNA genomes, and indeed even artistic.

Here it is in 3D!

  • Yes, the crazies are really crazy.
  • Secular humanism, in the sentimental clutches of Paul Kurtz.
  • Is BofA the next Lehman, going over the event horizon? Parts I, II, III
  • Bernanke's speech, dedicated to do-nothing-ism.
  • Shalizi on macroeconomic models, with link to a critique.
  • Diplomatically speaking, talking is OK.
  • Economic quotes of the week, from Bill Mitchell
"Our real world laboratory is providing priceless data upon which we can assess basic propositions that mainstream macroeconomics provides and which Modern Monetary Theory (MMT) contests. A nation cannot have a fiscal contraction expansion when all other spending is flat or going backwards. Britain is up against an impossible equation."
  • And, on the "believers in laissez-faire". A Kuhnian expired paradigm is on its last legs, waiting for its proponents to die off.
"One thing that is clear – the majority of these economists never have to carry the costs of their denial and retire on nice pensions. The same cannot be said for the victims of their arrogance and denial."

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